doxycycline responsive transactivator tet on 3g  (TaKaRa)


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    Structured Review

    TaKaRa doxycycline responsive transactivator tet on 3g
    MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of <t>Tet-On</t> 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .
    Doxycycline Responsive Transactivator Tet On 3g, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition"

    Article Title: Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2017.12.011

    MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of Tet-On 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .
    Figure Legend Snippet: MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of Tet-On 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .

    Techniques Used: Expressing, Electroporation, Cell Culture, Derivative Assay, Flow Cytometry, Cytometry, Labeling

    2) Product Images from "Star-PAP, a poly(A) polymerase, functions as a tumor suppressor in an orthotopic human breast cancer model"

    Article Title: Star-PAP, a poly(A) polymerase, functions as a tumor suppressor in an orthotopic human breast cancer model

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.199

    Star-PAP inhibits progression of breast cancer in vivo . ( a ) MDA-MB-468 Tet-On Star-PAP cells were induced with 200 ng/ml doxycycline for 24 h. Cell lysates were detected for Star-PAP and BIK by western blot. β -Tubulin was used as the loading control. ( b ) Loss of ΔΨ m in Tet-On Star-PAP cells was analyzed by JC-1 assay after treatment with doxycycline (left panel). Data from triplicate experiments were presented as mean±S.D. (right panel). ( c ) The same amount of Tet-On control and Star-PAP cells was separately incubated into the mammary fat pad of NOD/SCID mice, and one group of mice was supplied with diet containing 0.02% doxycycline immediately. Tumor volume was measured weekly and presented as mean±S.D.; n =8, ** P
    Figure Legend Snippet: Star-PAP inhibits progression of breast cancer in vivo . ( a ) MDA-MB-468 Tet-On Star-PAP cells were induced with 200 ng/ml doxycycline for 24 h. Cell lysates were detected for Star-PAP and BIK by western blot. β -Tubulin was used as the loading control. ( b ) Loss of ΔΨ m in Tet-On Star-PAP cells was analyzed by JC-1 assay after treatment with doxycycline (left panel). Data from triplicate experiments were presented as mean±S.D. (right panel). ( c ) The same amount of Tet-On control and Star-PAP cells was separately incubated into the mammary fat pad of NOD/SCID mice, and one group of mice was supplied with diet containing 0.02% doxycycline immediately. Tumor volume was measured weekly and presented as mean±S.D.; n =8, ** P

    Techniques Used: In Vivo, Multiple Displacement Amplification, Western Blot, Incubation, Mouse Assay

    Star-PAP sensitizes breast cancer cells to chemotherapy drugs. ( a ) Dose–response curves of Tet-On Star-PAP cells treated with cisplatin (left panel) and doxorubicin (right panel). Cell viability was measured by MTS assay; n =6, error bar denotes S.D. ( b ) Apoptosis of Tet-On Star-PAP cells treated with 1 μ M doxorubicin was analyzed in the absence or presence of doxycycline (left panel). Data from triplicate experiments are presented as mean±S.D. (right panel). ( c ) The same amount of Tet-On control and Star-PAP cells were incubated into the left and right mammary fat pad of NOD/SCID mice, respectively. Mice bearing xenografts were separated into two groups and treated with vehicle or doxorubicin, and diet containing doxycycline was supplied along with treatment. Tumor volume was measured weekly and presented as mean±S.D.; n =8, ** P
    Figure Legend Snippet: Star-PAP sensitizes breast cancer cells to chemotherapy drugs. ( a ) Dose–response curves of Tet-On Star-PAP cells treated with cisplatin (left panel) and doxorubicin (right panel). Cell viability was measured by MTS assay; n =6, error bar denotes S.D. ( b ) Apoptosis of Tet-On Star-PAP cells treated with 1 μ M doxorubicin was analyzed in the absence or presence of doxycycline (left panel). Data from triplicate experiments are presented as mean±S.D. (right panel). ( c ) The same amount of Tet-On control and Star-PAP cells were incubated into the left and right mammary fat pad of NOD/SCID mice, respectively. Mice bearing xenografts were separated into two groups and treated with vehicle or doxorubicin, and diet containing doxycycline was supplied along with treatment. Tumor volume was measured weekly and presented as mean±S.D.; n =8, ** P

    Techniques Used: MTS Assay, Incubation, Mouse Assay

    3) Product Images from "Receptor FGFRL1 does not promote cell proliferation but induces cell adhesion"

    Article Title: Receptor FGFRL1 does not promote cell proliferation but induces cell adhesion

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2016.2601

    Effect of fibroblast growth factor receptor-like protein 1 (FGFRL1) expression on cell proliferation. Stable HEK-TetOn clones were cultivated in the presence or absence of doxycycline in 24-well plates. Cell proliferation was measured over the course of five days with the hexosaminidase assay. No effect on cell proliferation was observed by the presence of FGFRL1 or FGFRL1ΔC. Standard errors are given at some time-points where meaningful (n=3).
    Figure Legend Snippet: Effect of fibroblast growth factor receptor-like protein 1 (FGFRL1) expression on cell proliferation. Stable HEK-TetOn clones were cultivated in the presence or absence of doxycycline in 24-well plates. Cell proliferation was measured over the course of five days with the hexosaminidase assay. No effect on cell proliferation was observed by the presence of FGFRL1 or FGFRL1ΔC. Standard errors are given at some time-points where meaningful (n=3).

    Techniques Used: Expressing, Clone Assay, Hexosaminidase Assay

    Effect of fibroblast growth factor receptor-like protein 1 (FGFRL1) on ERK phosphorylation. FGFRL1 inducible HEK-TetOn cells were cultivated in multi-well plates, starved overnight in medium lacking fetal bovine serum and then stimulated for 0–60 min as indicated with human FGF2 (15 ng/ml). Cells were lysed with hot SDS sample buffer. Cellular proteins were resolved on polyacrylamide gels and processed for western blotting with antibodies against phosphorylated ERK1/2 (ERK-p). The blots were stripped and reprobed with antibodies against total ERK (ERK-tot). An experiment conducted with clone K24F is depicted in panel A. Panel B shows an experiment with clone K17F. However in this case, the cells had been transfected - prior to starvation and FGF2 stimulation - with a full-length clone for human FGFR1 to increase signaling. ERK1/2 phosphorylation was extremely low before stimulation, but clearly visible after stimulation with FGF2. No difference in ERK phosphorylation was observed between doxycycline-induced and uninduced cells.
    Figure Legend Snippet: Effect of fibroblast growth factor receptor-like protein 1 (FGFRL1) on ERK phosphorylation. FGFRL1 inducible HEK-TetOn cells were cultivated in multi-well plates, starved overnight in medium lacking fetal bovine serum and then stimulated for 0–60 min as indicated with human FGF2 (15 ng/ml). Cells were lysed with hot SDS sample buffer. Cellular proteins were resolved on polyacrylamide gels and processed for western blotting with antibodies against phosphorylated ERK1/2 (ERK-p). The blots were stripped and reprobed with antibodies against total ERK (ERK-tot). An experiment conducted with clone K24F is depicted in panel A. Panel B shows an experiment with clone K17F. However in this case, the cells had been transfected - prior to starvation and FGF2 stimulation - with a full-length clone for human FGFR1 to increase signaling. ERK1/2 phosphorylation was extremely low before stimulation, but clearly visible after stimulation with FGF2. No difference in ERK phosphorylation was observed between doxycycline-induced and uninduced cells.

    Techniques Used: Western Blot, Transfection

    Effect of fibroblast growth factor receptor-like protein 1 (FGFRL1) on cell adhesion. FGFRL1 inducible HEK-TetOn cells were seeded into bacterial (non-tissue culture) petri dishes and cultivated for 10–15 h in the absence (minus) or presence (plus) of the inducer doxycycline. Note that expression of FGRL1, in its full-length form as well as its truncated form, promoted cell-cell adhesion and cell clustering. In sharp contrast, most cells remained in their single-cell state in the absence of FGFRL1 within this short period of time. A control with original HEK-TetOn cells (prior to transfection with FGFRL1 constructs) did not show any differences between the two conditions.
    Figure Legend Snippet: Effect of fibroblast growth factor receptor-like protein 1 (FGFRL1) on cell adhesion. FGFRL1 inducible HEK-TetOn cells were seeded into bacterial (non-tissue culture) petri dishes and cultivated for 10–15 h in the absence (minus) or presence (plus) of the inducer doxycycline. Note that expression of FGRL1, in its full-length form as well as its truncated form, promoted cell-cell adhesion and cell clustering. In sharp contrast, most cells remained in their single-cell state in the absence of FGFRL1 within this short period of time. A control with original HEK-TetOn cells (prior to transfection with FGFRL1 constructs) did not show any differences between the two conditions.

    Techniques Used: Expressing, Transfection, Construct

    Generation of HEK-TetOn cell lines with tetracycline-inducible fibroblast growth factor receptor-like protein 1 (FGFRL1) expression. (A) Schematic representation of the two FGFRL1 constructs that were stably transfected into HEK-TetOn cells. FGFRL1 represents the full-length, wild-type receptor, while FGFRL1ΔC contains a truncated C-terminal domain lacking the histidine-rich sequence and the tandem tyrosine motif. The two motifs are known to control the retention time of the receptor at the cell membrane. Hygromycin resistant cell clones were analyzed by northern blotting for inducible FGFRL1 expression. (B) Three representative full-length clones and (C) three representative C-terminally truncated clones are shown before and after induction with 1 µ g/ml doxycycline. As a loading control, the 28S ribosomal RNA stained with ethidium bromide is included.
    Figure Legend Snippet: Generation of HEK-TetOn cell lines with tetracycline-inducible fibroblast growth factor receptor-like protein 1 (FGFRL1) expression. (A) Schematic representation of the two FGFRL1 constructs that were stably transfected into HEK-TetOn cells. FGFRL1 represents the full-length, wild-type receptor, while FGFRL1ΔC contains a truncated C-terminal domain lacking the histidine-rich sequence and the tandem tyrosine motif. The two motifs are known to control the retention time of the receptor at the cell membrane. Hygromycin resistant cell clones were analyzed by northern blotting for inducible FGFRL1 expression. (B) Three representative full-length clones and (C) three representative C-terminally truncated clones are shown before and after induction with 1 µ g/ml doxycycline. As a loading control, the 28S ribosomal RNA stained with ethidium bromide is included.

    Techniques Used: Expressing, Construct, Stable Transfection, Transfection, Sequencing, Clone Assay, Northern Blot, Staining

    4) Product Images from "Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression"

    Article Title: Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116373

    Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems. (a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1μg/ml Doxycycline for 24hrs. (b) Expanded background luciferase activities in the absence of Doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet-On 3G to Doxycycline using a Tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of Doxycycline. Data show fold inductions of mean relative luciferase for Doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer-LSL) Doxycycline-inducible destination plasmids.
    Figure Legend Snippet: Construction of sensitive, low background Dox-inducible lentiviral and Flp-In systems. (a) Tetracycline-responsive luciferase reporter gene assays in HEK293T cells using Tet-On variants with (black) or without (grey) 1μg/ml Doxycycline for 24hrs. (b) Expanded background luciferase activities in the absence of Doxycycline from (a). Data are mean relative luciferase activities ± SEM of 3 independent experiments. (c) The sensitivity of Tet-On or Tet-On 3G to Doxycycline using a Tetracycline-responsive luciferase reporter gene in HEK293T cells in the presence (black) or absence (grey) of indicated concentrations of Doxycycline. Data show fold inductions of mean relative luciferase for Doxycycline treated versus non-treated cells, ± SEM of 3 independent experiments. (d) Schematic of vectors for fluorescent miR30c donor plasmids containing dsRed, dnucTomato, or eYFP. (e) Schematic of vectors for Lentiviral (LVTPTIG and LVTPTIP) and FLP-Inducer (Col1a1-FLP-Inducer and Col1a1-FLP-Inducer-LSL) Doxycycline-inducible destination plasmids.

    Techniques Used: Luciferase

    Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons. (a) Vector diagram of constitutively expressed (Tet-On 3G and eGFP) and Doxycycline-inducible (dsRed (cDNA), Tet-On 3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1μg/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP-dsRed-shGL3 and selected with Puromycin in the absence (red) or presence of 1μg/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4–3xFlag and treated with (+) or without (-) 1 μg/ml Doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1μg/ml Doxycycline for 24hrs.
    Figure Legend Snippet: Lentiviral LVTPT system allows for inducible cDNA and shRNA expression in stable cell lines and primary neurons. (a) Vector diagram of constitutively expressed (Tet-On 3G and eGFP) and Doxycycline-inducible (dsRed (cDNA), Tet-On 3G and eGFP) components of the LVTPT system (b) dsRed (left), eGFP (middle), or merged (right) fluorescence images of HEK293T cells infected with LVTPTIG-dsRed virus in the absence (lower panel) or presence (upper panel) of 1μg/ml Doxycycline for 24hrs (c) FACS analysis of dsRed expressing cells in uninfected (orange) or HEK293T cells stably infected with LVTPTIP-dsRed-shGL3 and selected with Puromycin in the absence (red) or presence of 1μg/ml Doxycycline for 24 (green) or 48 (blue) hrs. (d) LVTPTIP elicits effective RNAi in HEK293T cells. HEK293T cells stably infected with LVTPTIP-dsRED-shGL3 (negative control), LVTPTIP-dsRED-shNPAS4 2182 (impotent shRNA), or LVTPTIP-dsRED-shNPAS4 275134 (potent shRNA) were transfected with expression vector pEFBOS-NPAS4–3xFlag and treated with (+) or without (-) 1 μg/ml Doxycycline for 72 hrs. Whole cell extracts were analysed for NPAS4 (anti-Flag) and tubulin levels by immunoblot. (e) dsRed fluorescence images of embryonic day 16 mouse cortical neurons cultured in vitro and infected with concentrated LVTPT-dsRed-shGL3 virus and treated with (right panel) or without (left panel) 1μg/ml Doxycycline for 24hrs.

    Techniques Used: shRNA, Expressing, Stable Transfection, Plasmid Preparation, Fluorescence, Infection, FACS, Negative Control, Transfection, Cell Culture, In Vitro

    5) Product Images from "Transient, Inducible, Placenta-Specific Gene Expression in Mice"

    Article Title: Transient, Inducible, Placenta-Specific Gene Expression in Mice

    Journal: Endocrinology

    doi: 10.1210/en.2012-1556

    Generation of transgenic mice ubiquitously expressing Tet-On 3G protein. A, Strategy for Tet-On 3G transgenic mouse production. Zygotes from H11P3 FVB mice were used for microinjection of the pattB-Tet-On 3G plasmid and φC31 integrase mRNA. Recombination
    Figure Legend Snippet: Generation of transgenic mice ubiquitously expressing Tet-On 3G protein. A, Strategy for Tet-On 3G transgenic mouse production. Zygotes from H11P3 FVB mice were used for microinjection of the pattB-Tet-On 3G plasmid and φC31 integrase mRNA. Recombination

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Plasmid Preparation

    6) Product Images from "The Transcriptional Co-Repressor Myeloid Translocation Gene 16 Inhibits Glycolysis and Stimulates Mitochondrial Respiration"

    Article Title: The Transcriptional Co-Repressor Myeloid Translocation Gene 16 Inhibits Glycolysis and Stimulates Mitochondrial Respiration

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068502

    Requirement of Nervy Homology Region (NHR) 2 and 3 for MTG16–mediated gene expression inhibition. The Tet-On 3G doxycycline (DOX)–dependent gene expression system was used to regulate the expression of MTG16 NHR1-4 deletions inserted under the control of a TRE3G promoter in B-lymphoblastoid Raji target cells. A . Scheme of MTG16 with NHRs indicated. Deletions of the individual NHRs indicated created MTG16 ΔNHR1, MTG16 ΔNHR2, MTG16 ΔNHR3 and MTG16 ΔNHR4. B . Expression of mRNA for MTG16 NHR deletion 1-4 and full-length MTG16 . Cells were incubated with 20 ng/ml doxycycline for 24 h to induce gene expression before isolation of RNA and examination with PCR as described in Methods . The mRNA of different NHR deletions showed an expected size. C . Doxycycline (Dox) induction of MTG16 NHR1-4 deletions. Cells were incubated with 20 ng/ml doxycycline for 24 h and MTG16 in cell lysates was probed by Western blot analysis. All MTG16 NHR deletions showed expected lower size than full–length MTG16 from doxycycline–induced Raji/MTG16 Tet-On 3G cells (Raji Dox+). The polyclonal anti-MTG antibody used in Western blotting was raised against amino acids 31–250 of MTG8 [41] and recognizes wildtype MTG16 as well as the deletion mutants used. D . Effect of MTG16 NHR1-4 deletions on RNA expression of selected genes the expression of which was inhibited by MTG16. Cells were incubated without doxycycline (open bars) or with 20 ng/ml doxycycline (closed bars) for 24 h followed by examination of gene expression by RT-qPCR. Deletion of NHR2 or 3 abolished MTG16–mediated gene repression. Data in C are represented as means±SEM for n = 3 and were compared by the two-way ANOVA followed by the Bonferroni post-hoc test (*p
    Figure Legend Snippet: Requirement of Nervy Homology Region (NHR) 2 and 3 for MTG16–mediated gene expression inhibition. The Tet-On 3G doxycycline (DOX)–dependent gene expression system was used to regulate the expression of MTG16 NHR1-4 deletions inserted under the control of a TRE3G promoter in B-lymphoblastoid Raji target cells. A . Scheme of MTG16 with NHRs indicated. Deletions of the individual NHRs indicated created MTG16 ΔNHR1, MTG16 ΔNHR2, MTG16 ΔNHR3 and MTG16 ΔNHR4. B . Expression of mRNA for MTG16 NHR deletion 1-4 and full-length MTG16 . Cells were incubated with 20 ng/ml doxycycline for 24 h to induce gene expression before isolation of RNA and examination with PCR as described in Methods . The mRNA of different NHR deletions showed an expected size. C . Doxycycline (Dox) induction of MTG16 NHR1-4 deletions. Cells were incubated with 20 ng/ml doxycycline for 24 h and MTG16 in cell lysates was probed by Western blot analysis. All MTG16 NHR deletions showed expected lower size than full–length MTG16 from doxycycline–induced Raji/MTG16 Tet-On 3G cells (Raji Dox+). The polyclonal anti-MTG antibody used in Western blotting was raised against amino acids 31–250 of MTG8 [41] and recognizes wildtype MTG16 as well as the deletion mutants used. D . Effect of MTG16 NHR1-4 deletions on RNA expression of selected genes the expression of which was inhibited by MTG16. Cells were incubated without doxycycline (open bars) or with 20 ng/ml doxycycline (closed bars) for 24 h followed by examination of gene expression by RT-qPCR. Deletion of NHR2 or 3 abolished MTG16–mediated gene repression. Data in C are represented as means±SEM for n = 3 and were compared by the two-way ANOVA followed by the Bonferroni post-hoc test (*p

    Techniques Used: Expressing, Inhibition, Incubation, Isolation, Polymerase Chain Reaction, Western Blot, RNA Expression, Quantitative RT-PCR

    Doxycycline-induced expression of MTG16 in Raji/MTG16 Tet-On 3G cells. The Tet-On 3G doxycycline (DOX)–dependent gene expression system was used to regulate the expression of MTG16 inserted under the control of a TRE3G promoter in B-lymphoblastoid Raji target cells (Raji/MTG16 Tet-On 3G cells). A . MTG16 transcript induction with doxycycline. MTG16 mRNA is produced within 1 h of incubation with 20 ng/ml doxycycline as shown by RT-qPCR. B . Dose–response relationship for doxycycline induction of MTG16. MTG16 Tet-On 3G cells were incubated with various concentrations of doxycycline for 24 h and cell lysates were probed with anti-MTG by Western blot analysis for protein detection. Close to maximal MTG16 expression was obtained with 10 ng/ml of doxycycline. C . Time course for doxycycline induction of MTG16. Cells were incubated with 10 ng/ml doxycycline for various time periods and cell lysates were probed with anti-MTG by Western blot analysis. MTG16 protein was detected within 3 to 4 h. D . Cell cycle analysis of doxycycline–induced cells. Cells were incubated without (no DOX, blue) or with 20 ng/ml doxycycline (DOX, red) at 2×10 5 /ml up to 72 h and cell cycle analyses were performed by flow cytometry at intervals indicated. Representative cell cycle analyses are shown. Cell cycle distribution is also shown as percentage of cells in each phase. E . Inhibition of proliferation in doxycycline–induced MTG16 Tet-On cells. Proliferation curves are shown for MTG16 Tet-On cells incubated without doxycycline (blue), MTG16 Tet-On cells incubated with 20 ng/ml doxycycline (red) and control Raji cells expressing both EF1α-Tet3G and empty pTRE3G (without MTG16 ) incubated with 20 ng/ml doxycycline (black). MTG16 Tet-On cells incubated with doxycycline exhibited decreased proliferation compared to control cells. Cell viability was approximately 90% in all experiments and data are given for viable cells. Data are represented as means±SEM for (A) n = 3, (D) n = 3 and (E) n = 3 and were compared by the two-way ANOVA followed by Bonferroni (D) or the Tukey's multiple comparison post-hoc tests (E) (*p
    Figure Legend Snippet: Doxycycline-induced expression of MTG16 in Raji/MTG16 Tet-On 3G cells. The Tet-On 3G doxycycline (DOX)–dependent gene expression system was used to regulate the expression of MTG16 inserted under the control of a TRE3G promoter in B-lymphoblastoid Raji target cells (Raji/MTG16 Tet-On 3G cells). A . MTG16 transcript induction with doxycycline. MTG16 mRNA is produced within 1 h of incubation with 20 ng/ml doxycycline as shown by RT-qPCR. B . Dose–response relationship for doxycycline induction of MTG16. MTG16 Tet-On 3G cells were incubated with various concentrations of doxycycline for 24 h and cell lysates were probed with anti-MTG by Western blot analysis for protein detection. Close to maximal MTG16 expression was obtained with 10 ng/ml of doxycycline. C . Time course for doxycycline induction of MTG16. Cells were incubated with 10 ng/ml doxycycline for various time periods and cell lysates were probed with anti-MTG by Western blot analysis. MTG16 protein was detected within 3 to 4 h. D . Cell cycle analysis of doxycycline–induced cells. Cells were incubated without (no DOX, blue) or with 20 ng/ml doxycycline (DOX, red) at 2×10 5 /ml up to 72 h and cell cycle analyses were performed by flow cytometry at intervals indicated. Representative cell cycle analyses are shown. Cell cycle distribution is also shown as percentage of cells in each phase. E . Inhibition of proliferation in doxycycline–induced MTG16 Tet-On cells. Proliferation curves are shown for MTG16 Tet-On cells incubated without doxycycline (blue), MTG16 Tet-On cells incubated with 20 ng/ml doxycycline (red) and control Raji cells expressing both EF1α-Tet3G and empty pTRE3G (without MTG16 ) incubated with 20 ng/ml doxycycline (black). MTG16 Tet-On cells incubated with doxycycline exhibited decreased proliferation compared to control cells. Cell viability was approximately 90% in all experiments and data are given for viable cells. Data are represented as means±SEM for (A) n = 3, (D) n = 3 and (E) n = 3 and were compared by the two-way ANOVA followed by Bonferroni (D) or the Tukey's multiple comparison post-hoc tests (E) (*p

    Techniques Used: Expressing, Produced, Incubation, Quantitative RT-PCR, Western Blot, Cell Cycle Assay, Flow Cytometry, Cytometry, Inhibition

    7) Product Images from "FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells"

    Article Title: FER-mediated phosphorylation and PIK3R2 recruitment on IRS4 promotes AKT activation and tumorigenesis in ovarian cancer cells

    Journal: eLife

    doi: 10.7554/eLife.76183

    OVCAR-5 cells treated with TAE684 showed a dose-dependent growth inhibition. ( A ) HEK293FT cells were transiently transfected with pLV-FER, followed by TAE684 treatment with a series of concentrations for 16 hr, as indicated. The phosphorylation level of tyrosine 402 of FER was detected by immunoblotting with specific anti-pY402-FER. Total FER in whole cell lysate samples were also probed as loading control. IC 50 was calculated with dose-response nonlinear regression drawn by GraphPad Prism ( Figure 6—figure supplement 2—source data 1 ). ( B ) Immunoblotting of FES with four ovarian carcinoma-derived cell lines and one myeloid leukemia cell HL-60 to demonstrate no expression level of FES in these ovarian carcinoma-derived cell lines ( Figure 6—figure supplement 2—source data 2 ). ( C ) Cell proliferation was assessed in OVCAR-5 cells following incubation with increasing concentration of TAE684 for 48 hr using the CellTiter-Glo luminescent cell viability assay. ( D ) Cell proliferation was assessed in OVCAR-5 cells following incubation with increasing concentration of TAE684 for 72 hr using the CellTiter-Glo luminescent cell viability assay. Original images for Western blots in Figure 6—figure supplement 2A . Original images for Western blots in Figure 6—figure supplement 2B .
    Figure Legend Snippet: OVCAR-5 cells treated with TAE684 showed a dose-dependent growth inhibition. ( A ) HEK293FT cells were transiently transfected with pLV-FER, followed by TAE684 treatment with a series of concentrations for 16 hr, as indicated. The phosphorylation level of tyrosine 402 of FER was detected by immunoblotting with specific anti-pY402-FER. Total FER in whole cell lysate samples were also probed as loading control. IC 50 was calculated with dose-response nonlinear regression drawn by GraphPad Prism ( Figure 6—figure supplement 2—source data 1 ). ( B ) Immunoblotting of FES with four ovarian carcinoma-derived cell lines and one myeloid leukemia cell HL-60 to demonstrate no expression level of FES in these ovarian carcinoma-derived cell lines ( Figure 6—figure supplement 2—source data 2 ). ( C ) Cell proliferation was assessed in OVCAR-5 cells following incubation with increasing concentration of TAE684 for 48 hr using the CellTiter-Glo luminescent cell viability assay. ( D ) Cell proliferation was assessed in OVCAR-5 cells following incubation with increasing concentration of TAE684 for 72 hr using the CellTiter-Glo luminescent cell viability assay. Original images for Western blots in Figure 6—figure supplement 2A . Original images for Western blots in Figure 6—figure supplement 2B .

    Techniques Used: Inhibition, Transfection, Derivative Assay, Expressing, Incubation, Concentration Assay, Cell Viability Assay, Western Blot

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  • 95
    TaKaRa doxycycline responsive transactivator tet on 3g
    MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of <t>Tet-On</t> 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .
    Doxycycline Responsive Transactivator Tet On 3g, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    doxycycline responsive transactivator tet on 3g - by Bioz Stars, 2022-05
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    93
    TaKaRa hela tet on cells
    <t>HeLa</t> <t>Tet-on</t> cells expressing H-2K b were nucleofected with 4×100 nM siRNA. Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K b /S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K b cells pulsed for 2 h with 10 −8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K d stained with Abs 1, 2 and 3. One of two experiments is shown.
    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
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    Image Search Results


    MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of Tet-On 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .

    Journal: Stem Cell Reports

    Article Title: Expandable Arterial Endothelial Precursors from Human CD34+ Cells Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal Transition

    doi: 10.1016/j.stemcr.2017.12.011

    Figure Lengend Snippet: MYCN and SOX17 Induce and Expand Endothelial Precursors (A and B) Experimental approach. (A) Vectors used. Vectors were integrated in cells by the PiggyBac transposase. The promoter EF1α drives constitutive expression of Tet-On 3G . In the presence of doxycycline, Tet-On 3G binds the TRE3G promoter, upregulating expression of MYCN and SOX17 (encoded on separate vectors). (B) The three vectors from (A) were introduced by electroporation into human CD34 + cells (cultured for two days prior to electroporation). The electroporated cells were then cultured in the presence of doxycycline to induce colony formation and expansion. (C) Example colonies arising 9 days after electroporation as described in (B). Phase-contrast images. Scale bars, 400 μm. (D) Efficiencies of colony formation after 9 days. The boxes indicate the maximum to minimum efficiencies from at least two independent experiments; the horizontal lines within the boxes indicate the means. CB, cord blood; ABM, adult bone marrow. The range of the ages of the adult bone marrow donors is provided in years. (E) Growth curve, results are the average ± SD from six independent cell lines, three derived from cord blood and three derived from adult bone marrow. (F) Example phase-contrast images of endothelial precursor cell lines. Scale bars, 400 μm. (G and H) Cell lines were maintained in culture by the ectopic expression of MYCN and SOX17 (100 ng/mL doxycycline) and matured by downregulating the factors for 4 days (10 or 0 ng/mL doxycycline). 293T cells served as negative controls. Results are from two independent cell lines, one derived from cord blood and the other derived from adult bone marrow. (G) Analysis of the endothelial markers CDH5 and PECAM1 by flow cytometry. The number of days indicates the time in culture from the induction of the ectopic expression of MYCN and SOX17 . (H) Analysis of the uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) by flow cytometry. .

    Article Snippet: A cassette encoding the doxycycline-responsive transactivator Tet-On 3G (expressed by the constitutively active elongation factor 1 alpha (EF1α) promoter, Clontech) was also introduced into a PiggyBac vector.

    Techniques: Expressing, Electroporation, Cell Culture, Derivative Assay, Flow Cytometry, Cytometry, Labeling

    HeLa Tet-on cells expressing H-2K b were nucleofected with 4×100 nM siRNA. Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K b /S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K b cells pulsed for 2 h with 10 −8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K d stained with Abs 1, 2 and 3. One of two experiments is shown.

    Journal: PLoS ONE

    Article Title: No Major Role for Insulin-Degrading Enzyme in Antigen Presentation by MHC Molecules

    doi: 10.1371/journal.pone.0088365

    Figure Lengend Snippet: HeLa Tet-on cells expressing H-2K b were nucleofected with 4×100 nM siRNA. Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K b /S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K b cells pulsed for 2 h with 10 −8 M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K d stained with Abs 1, 2 and 3. One of two experiments is shown.

    Article Snippet: HeLa Tet-on cells (Clontech) stably transfected with H-2Kb (Hsu et al., submitted) were nucleofected with 4×100 nM siRNA.

    Techniques: Expressing, Labeling, Staining

    Forward trafficking of Cx43 to the plasma membrane is reduced during oxidative stress. ( A ) Timeline of Tet-inducible Cx43-EYFP trafficking assay. A tetracycline-inducible clonal HeLa cell line expressing Cx43-EYFP was induced with 2 μg/ml doxycycline 2.5 hours prior to imaging with TIRFm in the presence or absence of 200 μM H 2 O 2 , which was added 30 minutes prior to imaging. ( B ) TIRFm visualization of Cx43-EYFP delivery. TIRFm detection of Cx43-EYFP at 150, 195, and 240 minutes after the addition of doxycycline in the presence or absence of H 2 O 2 . White lines outline cell cortex. ( C ) Comparison of widefield epifluorescence and TIRFm detection of Cx43-EYFP at 240 minutes after the addition of doxycycline, showing that cells contain comparable levels of Cx43-EYFP. ( D ) Quantification of TIRFm-detectable Cx43-EYFP surface intensity. Error bars represent SEM. ( E ) Total and surface N-cadherin and Cx43-EYFP levels following total surface protein biotinylation and pulldown through neutravidin. Input lysates and pulldowns of biotinylated surface proteins were subjected to SDS-PAGE on the same gel. Original magnification, ×100. Scale bars: 10 μm. Values represent mean ± SEM.

    Journal: The Journal of Clinical Investigation

    Article Title: Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

    doi: 10.1172/JCI39740

    Figure Lengend Snippet: Forward trafficking of Cx43 to the plasma membrane is reduced during oxidative stress. ( A ) Timeline of Tet-inducible Cx43-EYFP trafficking assay. A tetracycline-inducible clonal HeLa cell line expressing Cx43-EYFP was induced with 2 μg/ml doxycycline 2.5 hours prior to imaging with TIRFm in the presence or absence of 200 μM H 2 O 2 , which was added 30 minutes prior to imaging. ( B ) TIRFm visualization of Cx43-EYFP delivery. TIRFm detection of Cx43-EYFP at 150, 195, and 240 minutes after the addition of doxycycline in the presence or absence of H 2 O 2 . White lines outline cell cortex. ( C ) Comparison of widefield epifluorescence and TIRFm detection of Cx43-EYFP at 240 minutes after the addition of doxycycline, showing that cells contain comparable levels of Cx43-EYFP. ( D ) Quantification of TIRFm-detectable Cx43-EYFP surface intensity. Error bars represent SEM. ( E ) Total and surface N-cadherin and Cx43-EYFP levels following total surface protein biotinylation and pulldown through neutravidin. Input lysates and pulldowns of biotinylated surface proteins were subjected to SDS-PAGE on the same gel. Original magnification, ×100. Scale bars: 10 μm. Values represent mean ± SEM.

    Article Snippet: HeLa Tet-ON cells were purchased from Clontech and maintained in fully supplemented DMEM containing 500 μg/ml Geneticin (Invitrogen).

    Techniques: Expressing, Imaging, SDS Page

    Flow cytometric analysis of cell cycle progression of pRetro-flag-RUNX3 stable HeLa Tet-On cell lines after release from thymidine block. ( A ) Histogram showing cell cycle profiles of the indicated p-RetroX-Flag-RUNX3 cell constructs at various time points

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

    Article Title: Aurora kinase-induced phosphorylation excludes transcription factor RUNX from the chromatin to facilitate proper mitotic progression

    doi: 10.1073/pnas.1523157113

    Figure Lengend Snippet: Flow cytometric analysis of cell cycle progression of pRetro-flag-RUNX3 stable HeLa Tet-On cell lines after release from thymidine block. ( A ) Histogram showing cell cycle profiles of the indicated p-RetroX-Flag-RUNX3 cell constructs at various time points

    Article Snippet: HeLa Tet-On cell line (Clontech) was infected with the constructs and selected with 0.5 µg/mL puromycin.

    Techniques: Blocking Assay, Construct