tet on 3g inducible expression system  (TaKaRa)


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    Structured Review

    TaKaRa tet on 3g inducible expression system
    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 <t>expression</t> 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 .
    Tet On 3g Inducible Expression System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tet on 3g inducible expression system/product/TaKaRa
    Average 95 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    tet on 3g inducible expression system - by Bioz Stars, 2022-08
    95/100 stars

    Images

    1) 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

    2) Product Images from "The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes"

    Article Title: The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes

    Journal: Communications Biology

    doi: 10.1038/s42003-022-03377-x

    Endogenous mitofusins locate on the mitochondrion-peroxisome contacting sites. a Immunofluorescence images of endogenous MFN2. Confocal microscopy analysis of HeLa cells expressing COX4-EGFP (mitochondria), immunostained for MFN2 (Alexa Fluor 555) and peroxisomal protein ABCD3 (Alexa Fluor 647). Scale bars: 5 μm. b The zoomed-in images of the white box in ( a ). White arrows point the places where ABCD3 overlaps with MFN. Scale bars: 1 μm. c Histograms display measured fluorescence intensity along the white line in the merge panels in b , with the cyan line represents mitochondria, the magenta line represents endogenous MFN2, and the red line represents peroxisome. d – f Immunofluorescence images and analysis of endogenous MFN1. Similar experiments were performed as in ( a – c ).
    Figure Legend Snippet: Endogenous mitofusins locate on the mitochondrion-peroxisome contacting sites. a Immunofluorescence images of endogenous MFN2. Confocal microscopy analysis of HeLa cells expressing COX4-EGFP (mitochondria), immunostained for MFN2 (Alexa Fluor 555) and peroxisomal protein ABCD3 (Alexa Fluor 647). Scale bars: 5 μm. b The zoomed-in images of the white box in ( a ). White arrows point the places where ABCD3 overlaps with MFN. Scale bars: 1 μm. c Histograms display measured fluorescence intensity along the white line in the merge panels in b , with the cyan line represents mitochondria, the magenta line represents endogenous MFN2, and the red line represents peroxisome. d – f Immunofluorescence images and analysis of endogenous MFN1. Similar experiments were performed as in ( a – c ).

    Techniques Used: Immunofluorescence, Confocal Microscopy, Expressing, Fluorescence

    Exogenous expression of MFNs enhance the PerMit Venus reporter signal. a Schematic for the constructs of Cyto-V(N), Mito-V(N), and Po-V(C). Cyto-V(N), HA tag fused to the N terminus of Venus with a linker composed of 4 × GGSG (indicated with blue box); Mito-V(N), Tom20 fused to the N terminus Venus with a HA tag and two linkers as indicated; Po-V(C), Myc tag and PEX26(residues 237-305) fused to the C terminus of Venus (residues 155-238). b Immuno-blots for Cyto-V(N), Mito-V(N), and Po-V(C). c Immunofluorescence images of Cyto-V(N), Mito-V(N), and Po-V(C) co-stained with mitochondrial COX4 and peroxisomal ABCD3 in HeLa cells. Scale bars, 5 μm. d Immunofluorescence images of Venus co-stained with mitochondrial COX4 and peroxisomal ABCD3 in PerMit Venus and control cells. Scale bars, 5 μm. e Immunofluorescence images of PerMit Venus cells with exogenously expressed MFNs. PerMit Venus cells were transfected with empty vector, MFN1-FLAG, or MFN2-FLAG plasmids for 36 h. FLAG (Alexa Fluor 568) and peroxisomal membrane protein PEX14 (Alexa Fluor 647) were immunostained. Scale bars, 5 μm. f Integrated density of ( e ), Vector, n = 67; MFN1-FLAG, n = 73; MFN2-FLAG, n = 72. *** p
    Figure Legend Snippet: Exogenous expression of MFNs enhance the PerMit Venus reporter signal. a Schematic for the constructs of Cyto-V(N), Mito-V(N), and Po-V(C). Cyto-V(N), HA tag fused to the N terminus of Venus with a linker composed of 4 × GGSG (indicated with blue box); Mito-V(N), Tom20 fused to the N terminus Venus with a HA tag and two linkers as indicated; Po-V(C), Myc tag and PEX26(residues 237-305) fused to the C terminus of Venus (residues 155-238). b Immuno-blots for Cyto-V(N), Mito-V(N), and Po-V(C). c Immunofluorescence images of Cyto-V(N), Mito-V(N), and Po-V(C) co-stained with mitochondrial COX4 and peroxisomal ABCD3 in HeLa cells. Scale bars, 5 μm. d Immunofluorescence images of Venus co-stained with mitochondrial COX4 and peroxisomal ABCD3 in PerMit Venus and control cells. Scale bars, 5 μm. e Immunofluorescence images of PerMit Venus cells with exogenously expressed MFNs. PerMit Venus cells were transfected with empty vector, MFN1-FLAG, or MFN2-FLAG plasmids for 36 h. FLAG (Alexa Fluor 568) and peroxisomal membrane protein PEX14 (Alexa Fluor 647) were immunostained. Scale bars, 5 μm. f Integrated density of ( e ), Vector, n = 67; MFN1-FLAG, n = 73; MFN2-FLAG, n = 72. *** p

    Techniques Used: Expressing, Construct, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation

    Exogenous expression of MFN induces peroxisome/mitochondrion clustering. Immunofluorescence images of overexpressed MFN-EGFP. HeLa cells were transfected with free EGFP ( a ) or EGFP fused MFN ( b ) plasmids for 36 h. Peroxisomal matrix protein catalase (Alexa Fluor 555) and peroxisomal membrane protein ABCD3 (Alexa Fluor 647) were immune-stained. Scale bars, 5 μm. c Peroxisomal membrane protein PEX14 (Alexa Fluor 555) and outer mitochondrial membrane protein Tom20 (Alexa Fluor 647) were immunostained with or without exogenously expressed MFNs. Scale bars, 5 μm. d Immuno-blots of overexpressed MFN1-EGFP and MFN2-EGFP. 1.5 µg plasmids were transfected into HeLa cells in one well in a six-well cell culture plate for 36 h and immunoblotted with indicated antibodies. e Immunofluorescence images of overexpressed MFN2-EGFP and other organelle markers. HeLa cells were transfected with MFN2-EGFP and immunostained for peroxisomal membrane protein ABCD3 (Alexa Fluor 647) and other organelle markers (Alexa Fluor 555): calnexin (endoplasmic reticulum), EEA1 (early endosome), GM130 (Golgi), and LAMP1 (lysosome). Scale bars, 5 μm. f Immunofluorescence images of overexpressed MFN1-EGFP and other organelle markers stained the same as in ( c ).
    Figure Legend Snippet: Exogenous expression of MFN induces peroxisome/mitochondrion clustering. Immunofluorescence images of overexpressed MFN-EGFP. HeLa cells were transfected with free EGFP ( a ) or EGFP fused MFN ( b ) plasmids for 36 h. Peroxisomal matrix protein catalase (Alexa Fluor 555) and peroxisomal membrane protein ABCD3 (Alexa Fluor 647) were immune-stained. Scale bars, 5 μm. c Peroxisomal membrane protein PEX14 (Alexa Fluor 555) and outer mitochondrial membrane protein Tom20 (Alexa Fluor 647) were immunostained with or without exogenously expressed MFNs. Scale bars, 5 μm. d Immuno-blots of overexpressed MFN1-EGFP and MFN2-EGFP. 1.5 µg plasmids were transfected into HeLa cells in one well in a six-well cell culture plate for 36 h and immunoblotted with indicated antibodies. e Immunofluorescence images of overexpressed MFN2-EGFP and other organelle markers. HeLa cells were transfected with MFN2-EGFP and immunostained for peroxisomal membrane protein ABCD3 (Alexa Fluor 647) and other organelle markers (Alexa Fluor 555): calnexin (endoplasmic reticulum), EEA1 (early endosome), GM130 (Golgi), and LAMP1 (lysosome). Scale bars, 5 μm. f Immunofluorescence images of overexpressed MFN1-EGFP and other organelle markers stained the same as in ( c ).

    Techniques Used: Expressing, Immunofluorescence, Transfection, Staining, Western Blot, Cell Culture

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    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
    https://www.bioz.com/result/doxycycline responsive transactivator tet on 3g/product/TaKaRa
    Average 95 stars, based on 1 article reviews
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    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

    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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2016.199

    Figure Lengend 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

    Article Snippet: For Tet-On inducible expression, Star-PAP was cloned into pLVX-TRE3G response vector (Clontech, Mountain View, CA, USA), and then the lentivirus production and infection was conducted according to the manufacturer's instructions.

    Techniques: 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

    Journal: Cell Death & Disease

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

    doi: 10.1038/cddis.2016.199

    Figure Lengend 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

    Article Snippet: For Tet-On inducible expression, Star-PAP was cloned into pLVX-TRE3G response vector (Clontech, Mountain View, CA, USA), and then the lentivirus production and infection was conducted according to the manufacturer's instructions.

    Techniques: MTS Assay, Incubation, Mouse Assay

    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).

    Journal: International Journal of Molecular Medicine

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

    doi: 10.3892/ijmm.2016.2601

    Figure Lengend 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).

    Article Snippet: Cell culture HEK-TetOn cells were purchased along with the advanced TetOn expression system from Clontech Laboratories (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France).

    Techniques: 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.

    Journal: International Journal of Molecular Medicine

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

    doi: 10.3892/ijmm.2016.2601

    Figure Lengend 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.

    Article Snippet: Cell culture HEK-TetOn cells were purchased along with the advanced TetOn expression system from Clontech Laboratories (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France).

    Techniques: 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.

    Journal: International Journal of Molecular Medicine

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

    doi: 10.3892/ijmm.2016.2601

    Figure Lengend 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.

    Article Snippet: Cell culture HEK-TetOn cells were purchased along with the advanced TetOn expression system from Clontech Laboratories (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France).

    Techniques: 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.

    Journal: International Journal of Molecular Medicine

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

    doi: 10.3892/ijmm.2016.2601

    Figure Lengend 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.

    Article Snippet: Cell culture HEK-TetOn cells were purchased along with the advanced TetOn expression system from Clontech Laboratories (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France).

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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0116373

    Figure Lengend 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.

    Article Snippet: Generation of lentiviral and RMCE constructs pCMV-Tet-On variant 16 (V16; V9I, F67S, R171K, F86Y, A209T)[ ] was kindly provided by A.T. Das, pCMV-Tet-On[ ], and Tet-On 3G (V10; F67S, R171K, F86Y, A209T)[ ] purchased from Clonetech.

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0116373

    Figure Lengend 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.

    Article Snippet: Generation of lentiviral and RMCE constructs pCMV-Tet-On variant 16 (V16; V9I, F67S, R171K, F86Y, A209T)[ ] was kindly provided by A.T. Das, pCMV-Tet-On[ ], and Tet-On 3G (V10; F67S, R171K, F86Y, A209T)[ ] purchased from Clonetech.

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