hek293t cells Search Results


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  • 99
    ATCC hek293t cells hek293t cells
    E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of <t>HEK293T</t> cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.
    Hek293t Cells Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1836 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher hek 293t cells hek293t cells
    E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of <t>HEK293T</t> cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.
    Hek 293t Cells Hek293t Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 5 article reviews
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    99
    ATCC cell culture hek293t cells
    FIN mediates the COX2/PGE 2 pathway by increasing the activity of COX2 promoter. The plasmid harboring a COX2 promoter fragment and a firefly luciferase (FL) gene (5ng/well separately) was transfected into <t>HEK293T</t> cells in a 24-well-plate and a CMV-Renilla luciferase (RL) plasmid (10ng/well) served as a transfection control. FIN (50μM) was treated for 6 h before harvesting the cells for dual-luciferase assay. Transfected cells treated with DMSO were set as negative control. Results are shown in mean ± SEM and representative of 3 independent experiments. **p
    Cell Culture Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 248 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 248 article reviews
    Price from $9.99 to $1999.99
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    99
    ATCC hek 293t cells hek293t cells
    IL-1β induces the expression of miR-31, which affects E-selectin abundance a. The monolayer of HUVEC endothelial cells was treated with IL-1β (20ng/ml) or TNFα (10ng/ml) for indicated hours. Western blotting monitored the expression of E-selectin and Actin used as loading control. b. The miR-31 level was measured by quantitative reverse transcription-PCR and the snRNA U6 was used as the normalization control. c. Endothelial cells were transfected with 30nM of either anti-miR-31 (+) or corresponding inhibitor negative control (−) before the addition of IL-1β. The Western blots shown are representative of three independent experiments. The endogenous GAPDH was used as loading control. d. Top: The representation of miR-31 base-pairing with either E-selectin wild-type (wt) or mutated (mut) 3′ UTR sequence. The nucleotides in the gray box represent the seed region of miR-31, region important for target interaction. Bottom: E-selectin 3′UTR mediated reporter assays. Vector expressing luciferase reporter under the regulation of either wild-type (black bars) or mutated (white bars) E-selectin 3′UTR were transfected into <t>HEK293T</t> cells. 48 hours after transfection, relative luciferase activities were measured. Another set of reporters transfected cells were also transfected with 30nM of either anti-miR-31 or corresponding inhibitor negative control (ctl) to further test the contribution of miR-31 in E-selectin regulation. The error bars shown in panel b and d represent standard errors of three and six independent experiments, respectively. The significance was analyzed using a Student's t -test (* p
    Hek 293t Cells Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATCC transfection hek293t cells
    Epilepsy‐associated mutations alter the function of the Nav1.2 channel. (a) Representative whole‐cell patch‐clamp traces of voltage‐dependent currents recorded from <t>HEK293T</t> cells transfected with either Nav1.2 wild‐type or mutant channels. Cells were held at −120 mV and sodium currents were evoked by a series of depolarizing pulses (100 ms) to potentials ranging from −80 mV to +90 mV in steps of 5 mV (inset). (b) Normalized I‒V curves of peak sodium current density (in pA/pF) versus voltage for WT and mutations. (c) Voltage dependence of activation obtained by plotting the normalized conductance against test potentials with equation G/G max = 1/(1 + exp(V 0.5 − V)/k, where the G max is the maximum conductance, V 0.5 is the half‐maximal activation potential and k is the slope factor. the curve is fitted to a Boltzmann function. (d) Steady state of fast inactivation of WT and mutant Nav1.2 channels. The voltage dependence of fast inactivation was assessed by applying a double‐pulse protocol: 500‐ms prepulses were applied from −150 to 0 mV in steps of 10 mV and followed by a test pulse to −10 mV (inset). The steady‐state fast inactivation curve was fitted by the Boltzmann equation (I/Imax = {1 + exp[(V − V 0.5 )/k]} − 1). (e) Window currents of WT the mutations. Activation curves (fraction of maximum conductance, G/G max ) and steady‐state inactivation (fraction of maximum current, I/Imax) of WT and the mutations are enlarged and plotted together to show the window currents. (f) The time course of recovery from fast inactivation. Recovery from fast inactivation was assessed by a two‐pulse recovery protocol with varying time intervals between a 500‐ms inactivating prepulse and a test pulse to −10 mV (inset). The time course of recovery from inactivation was fitted with a double‐exponential function to generate τ1 and τ2. All fitting results are listed in Table 1 . Data are presented as the mean ± SEM
    Transfection Hek293t Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 93/100, based on 364 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of HEK293T cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: E488Q mutation introduced into DD increases editing efficiency. ( A ) Schematic of the E488Q mutation in λN-DD or 4λN-DD. ( B ) Confocal images of HEK293T cells transfected with various components of SDRE. Scale bars = 12 μm. ( C ) Quantification of correction in transfections by both fluorescence (dark gray) and direct sequencing (light gray). All values for fluorescence correction are significantly different by ANOVA and Tukey's test. n = 200–800 cells, mean ± SEM. Technical duplicates for all fluorescence measurements yielded similar results (11.5 ± 0.1, 32 ± 0.2, 58.9 ± 2.1 and 65 ± 0.02 for λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively). Technical duplicates for RNA correction were as follows: 7, 27, 69 and 70 % for direct sequencing measurements of λN-DD 1boxB, λN-DD E488Q 1boxB, 4λN-DD 2boxB and 4λN-DD E488Q 2boxB, respectively.

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Mutagenesis, Transfection, Fluorescence, Sequencing

    Editing efficiencies of optimized SDRE system on A's in different neighboring contexts. ( A ) A's in different neighboring contexts were introduced into mCherry-eGFP and different 2boxB guide RNAs were designed to target them. These RNA guides were tested in HEK293T cells with either 4λN -DD or 4λN-DD E488Q. Results were tested only by direct sequencing of RT-PCR products. Experiments were done in duplicate. ( B ) Editing percentages of individual A's for the UAA N context with 4λN -DD and 4λN-DD E488Q. Rep 1 = repetition 1 and rep2 = repetition 2.

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: Editing efficiencies of optimized SDRE system on A's in different neighboring contexts. ( A ) A's in different neighboring contexts were introduced into mCherry-eGFP and different 2boxB guide RNAs were designed to target them. These RNA guides were tested in HEK293T cells with either 4λN -DD or 4λN-DD E488Q. Results were tested only by direct sequencing of RT-PCR products. Experiments were done in duplicate. ( B ) Editing percentages of individual A's for the UAA N context with 4λN -DD and 4λN-DD E488Q. Rep 1 = repetition 1 and rep2 = repetition 2.

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

    Increasing the number of λNs and boxBs improves editing. ( A i) A schematic showing the addition of N-terminal λN's. ( A ii) Reaction kinetics of λN-DD and 2λN-DD, purified from Pichia pastoris , were measured using mCherry-eGFP W58X in vitro using our standard 1boxB RNA guide. Rate constants, estimated from fits (see Materials and Methods section), were k obs = 0.1 ± 0.02 and 0.3 ± 0.05 min −1 for λN-DD and 2λN-DD, respectively. Experiments were performed at 35°C. Constructs with 1–5λNs connected to DD were generated and tested in HEK293T cells using the fluorescent reporter assay (dark gray) and by RT-PCR (light gray; A iii). Technical duplicates for all fluorescence measurements yielded similar results (11.6 ± 0.5, 19.2 ± 0.9, 32.1 ± 1.3, 38.5 ± 0.7 and 35.7 ± 1.3 % for 1–5λN-DD, respectively). Technical duplicates for RNA correction were as follows: 8, 27, 32, 48 and 41 % for direct sequencing measurements of 1–5λN-DD, respectively. ( B i) 1boxB and 2boxB RNA guides. With the 2boxB guide, the target A (red) is 19 nt 3′ to boxB 1 and 11 nt 5′ to boxB 2. ( B ii) The kinetics of editing using a 1boxB or 2BoxB guide was tested with purified λN-DD and mCherry-eGFP W58X in vitro . Temperature = 35°C. The data for 2boxB was fitted to a double exponential and the rate constants were k 1 obs = 1.54 ± 0.09 and k 2 obs = 0.05 ± 0.001. ( B iii) Editing efficiency of 2boxB guide was tested in HEK293T cells using 1–4λN-DD. As before, correction was estimated by both fluorescence (dark gray) and direct sequencing (light gray). All results were tested with a one-way ANOVA and Tukey's test. An asterisk indicates significance ( P

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: Increasing the number of λNs and boxBs improves editing. ( A i) A schematic showing the addition of N-terminal λN's. ( A ii) Reaction kinetics of λN-DD and 2λN-DD, purified from Pichia pastoris , were measured using mCherry-eGFP W58X in vitro using our standard 1boxB RNA guide. Rate constants, estimated from fits (see Materials and Methods section), were k obs = 0.1 ± 0.02 and 0.3 ± 0.05 min −1 for λN-DD and 2λN-DD, respectively. Experiments were performed at 35°C. Constructs with 1–5λNs connected to DD were generated and tested in HEK293T cells using the fluorescent reporter assay (dark gray) and by RT-PCR (light gray; A iii). Technical duplicates for all fluorescence measurements yielded similar results (11.6 ± 0.5, 19.2 ± 0.9, 32.1 ± 1.3, 38.5 ± 0.7 and 35.7 ± 1.3 % for 1–5λN-DD, respectively). Technical duplicates for RNA correction were as follows: 8, 27, 32, 48 and 41 % for direct sequencing measurements of 1–5λN-DD, respectively. ( B i) 1boxB and 2boxB RNA guides. With the 2boxB guide, the target A (red) is 19 nt 3′ to boxB 1 and 11 nt 5′ to boxB 2. ( B ii) The kinetics of editing using a 1boxB or 2BoxB guide was tested with purified λN-DD and mCherry-eGFP W58X in vitro . Temperature = 35°C. The data for 2boxB was fitted to a double exponential and the rate constants were k 1 obs = 1.54 ± 0.09 and k 2 obs = 0.05 ± 0.001. ( B iii) Editing efficiency of 2boxB guide was tested in HEK293T cells using 1–4λN-DD. As before, correction was estimated by both fluorescence (dark gray) and direct sequencing (light gray). All results were tested with a one-way ANOVA and Tukey's test. An asterisk indicates significance ( P

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Purification, In Vitro, Construct, Generated, Reporter Assay, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Sequencing

    Fluorescent reporter system for quantifying editing. ( A ) Cartoons of fluorescent protein constructs and plasmids used in this study. Full-length mCherry and eGFP were fused together and separated by a 2A peptide (black) followed by a FLAG epitope tag (grey). In addition, a version with a stop codon (U A G) inserted at position 58 of eGFP was made (W58X). These constructs were cloned into a vector driven by the CMV promoter, as was λN-DD. RNA guides were driven by a U6 promoter. ( B ) Confocal images of HEK293T cells transfected with different combinations of components are shown. Red, green, and DIC images are shown for the same field of cells. Pictures are taken 96 h post-transfection. Scale bars = 12 μm. Fluorescence correction was estimated to be 11 ± 0.05 %. ( C ) Sequences from RT-PCR products of corrected cells and cells transfected with mCherry-eGFP W58X alone. Asterisks indicate the target A. Editing percentage for experimental samples was estimated to be 11 %. ( D ) Estimates of editing efficiencies from fluorescence (dark gray) and direct sequencing (light grey) were compared at various days post-transfection. Fluorescence estimates were based on 200–800 cells per sample (mean ± SEM) and RT-PCR products came from the same samples. Technical duplicates for all fluorescence measurements yielded similar results (2.3 ± 0.3, 8.7 ± 1.5, 8.9 ± 1.5, and 12.9 ± 1.4 % for days 1, 2, 3, and 4, respectively). Technical duplicates for RNA correction based on direct sequencing of were as follows: 4, 6, 9 and 14 %, for days 1, 2, 3 and 4, respectively.

    Journal: Nucleic Acids Research

    Article Title: An efficient system for selectively altering genetic information within mRNAs

    doi: 10.1093/nar/gkw738

    Figure Lengend Snippet: Fluorescent reporter system for quantifying editing. ( A ) Cartoons of fluorescent protein constructs and plasmids used in this study. Full-length mCherry and eGFP were fused together and separated by a 2A peptide (black) followed by a FLAG epitope tag (grey). In addition, a version with a stop codon (U A G) inserted at position 58 of eGFP was made (W58X). These constructs were cloned into a vector driven by the CMV promoter, as was λN-DD. RNA guides were driven by a U6 promoter. ( B ) Confocal images of HEK293T cells transfected with different combinations of components are shown. Red, green, and DIC images are shown for the same field of cells. Pictures are taken 96 h post-transfection. Scale bars = 12 μm. Fluorescence correction was estimated to be 11 ± 0.05 %. ( C ) Sequences from RT-PCR products of corrected cells and cells transfected with mCherry-eGFP W58X alone. Asterisks indicate the target A. Editing percentage for experimental samples was estimated to be 11 %. ( D ) Estimates of editing efficiencies from fluorescence (dark gray) and direct sequencing (light grey) were compared at various days post-transfection. Fluorescence estimates were based on 200–800 cells per sample (mean ± SEM) and RT-PCR products came from the same samples. Technical duplicates for all fluorescence measurements yielded similar results (2.3 ± 0.3, 8.7 ± 1.5, 8.9 ± 1.5, and 12.9 ± 1.4 % for days 1, 2, 3, and 4, respectively). Technical duplicates for RNA correction based on direct sequencing of were as follows: 4, 6, 9 and 14 %, for days 1, 2, 3 and 4, respectively.

    Article Snippet: Editing assays in HEK293T cells HEK293T cells (CRL-11268 ATCC, Manassas, VA) were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10 % (v/v) fetal bovine serum, 1% penicillin–streptomycin solution, 1 mM sodium pyruvate, and 2 mM glutamine.

    Techniques: Construct, FLAG-tag, Clone Assay, Plasmid Preparation, Transfection, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Sequencing

    Gaussian fitting of the factor of viscosity for HEK293T cells. The cells were treated with (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL AgNPs for 24 h, respectively.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Gaussian fitting of the factor of viscosity for HEK293T cells. The cells were treated with (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL AgNPs for 24 h, respectively.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques:

    Images of DNA damages detected by comet assays for HEK293T cells treated with varying AgNPs concentrations for 24 h: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Images of DNA damages detected by comet assays for HEK293T cells treated with varying AgNPs concentrations for 24 h: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques:

    Cell viability of HEK293T cells tested by the MTT assay. HEK293T cells were treated with varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h. Following treatment with MTT reagents, viable cells were quantified by measuring the OD 490 of sample wells. * indicates p

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Cell viability of HEK293T cells tested by the MTT assay. HEK293T cells were treated with varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h. Following treatment with MTT reagents, viable cells were quantified by measuring the OD 490 of sample wells. * indicates p

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: MTT Assay

    Gene expression levels for HEK293T cells after exposure to varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h: (A) Bcl2-t , (B) Bclw , and (C) Bid . * indicates p

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Gene expression levels for HEK293T cells after exposure to varying AgNPs concentrations (0, 10, 20, and 40 μg/mL) for 24 h: (A) Bcl2-t , (B) Bclw , and (C) Bid . * indicates p

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: Expressing

    Biomechanical analysis of HEK293T cells in phosphate buffer solution (PBS) buffer. A sample force versus distance curve obtained on an untreated HEK293T cell shows the tip approach (red) and withdrawal (back). The energy involved in the indentation process includes two parts: elastic energy (green) and viscous energy (yellow).

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Biomechanical analysis of HEK293T cells in phosphate buffer solution (PBS) buffer. A sample force versus distance curve obtained on an untreated HEK293T cell shows the tip approach (red) and withdrawal (back). The energy involved in the indentation process includes two parts: elastic energy (green) and viscous energy (yellow).

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques:

    Representative images of HEK293T cells following exposure to varying AgNPs concentrations for 24 h, observed using an inverted optical microscope: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Representative images of HEK293T cells following exposure to varying AgNPs concentrations for 24 h, observed using an inverted optical microscope: (A) 0 μg/mL, (B) 10 μg/mL, (C) 20 μg/mL, and (D) 40 μg/mL.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: Microscopy

    Dose–response relationship for DNA damage in HEK293T cells after exposure to AgNPs measured using the comet assay. DNA damage in terms of (A) tail DNA% and (B) tail moment obtained at different AgNPs concentrations (0, 10, 20, and 40 μg/mL), fitted each with a linear regression line.

    Journal: ACS Omega

    Article Title: Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

    doi: 10.1021/acsomega.8b00608

    Figure Lengend Snippet: Dose–response relationship for DNA damage in HEK293T cells after exposure to AgNPs measured using the comet assay. DNA damage in terms of (A) tail DNA% and (B) tail moment obtained at different AgNPs concentrations (0, 10, 20, and 40 μg/mL), fitted each with a linear regression line.

    Article Snippet: 4.2 Cells Culture and Cell Viability Measurement HEK293T cells (American Type Culture Collection, CRL-11268, Shanghai, China) were cultured in Gibco Roswell Park Memorial Institute medium 1640 basic (1×) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Shanghai, China) and 1% penicillin–streptomycin solution (Beyotime, Jiangsu, China) at 37 °C in an incubator humidified with 5% CO2 atmosphere.

    Techniques: Single Cell Gel Electrophoresis

    Hyperphosphorylated UPF1 promotes CED complex formation. a IPs of FLAG-UPF1: either WT or one of its variants. HEK293T cells transiently expressing the indicated protein were treated with either DMSO or MG132 for 12 h before harvesting. The cell extracts were treated with RNase A and then subjected to IPs with the α-FLAG antibody; n = 3. b Quantitation of the Western blotting images presented in ( a ). Signal intensities of the Western blot bands were quantified. The levels of hyperphosphorylated UPF1 and coimmunopurified cellular proteins were normalized to the amounts of immunopurified FLAG-UPF1. The normalized levels obtained in the IP of FLAG-UPF1-WT were arbitrarily set to 1.0. Data are presented as mean values ± standard deviations (SD) and statistical significance; Two-tailed, equal-sample variance Student’s t test was carried out to calculate the P values. * P = 0.0102 and ** P

    Journal: Nature Communications

    Article Title: Nonsense-mediated mRNA decay factor UPF1 promotes aggresome formation

    doi: 10.1038/s41467-020-16939-6

    Figure Lengend Snippet: Hyperphosphorylated UPF1 promotes CED complex formation. a IPs of FLAG-UPF1: either WT or one of its variants. HEK293T cells transiently expressing the indicated protein were treated with either DMSO or MG132 for 12 h before harvesting. The cell extracts were treated with RNase A and then subjected to IPs with the α-FLAG antibody; n = 3. b Quantitation of the Western blotting images presented in ( a ). Signal intensities of the Western blot bands were quantified. The levels of hyperphosphorylated UPF1 and coimmunopurified cellular proteins were normalized to the amounts of immunopurified FLAG-UPF1. The normalized levels obtained in the IP of FLAG-UPF1-WT were arbitrarily set to 1.0. Data are presented as mean values ± standard deviations (SD) and statistical significance; Two-tailed, equal-sample variance Student’s t test was carried out to calculate the P values. * P = 0.0102 and ** P

    Article Snippet: Cell culture and chemical treatment HeLa and HEK293T cells (purchased from ATCC), and MEFs (a gift from Byung-Yoon Ahn, Korea University, Seoul, Korea) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Capricorn Scientific) containing 10% fetal bovine serum (Capricorn Scientific) and 1% penicillin/streptomycin (Capricorn Scientific).

    Techniques: Expressing, Quantitation Assay, Western Blot, Two Tailed Test

    Localization of UPF1 to the aggresome depends on its phosphorylation status. a Validation of UPF1 hyperphosphorylation. HEK293T cells were transiently transfected with a plasmid expressing either Myc-UPF1-WT (wild type) or one of its variants. Two days later, the cells were harvested. Next, the extract of cells was treated with RNase A before IPs and was subjected to IPs using the anti (α)-Myc antibody. The samples before and after IPs were analyzed by western blotting; n = 2. Source data are provided as a Source Data File. b Immunostaining of CFTR-ΔF508 (green) and either Myc-UPF1-WT or its variants (red). HeLa cells stably expressing CFTR-ΔF508 were transiently transfected with the indicated plasmid expressing either Myc-UPF1-WT or one of its variants. Immunostaining images are representative of three independently processed biological replicates. Scale bar, 10 μm; n = 3.

    Journal: Nature Communications

    Article Title: Nonsense-mediated mRNA decay factor UPF1 promotes aggresome formation

    doi: 10.1038/s41467-020-16939-6

    Figure Lengend Snippet: Localization of UPF1 to the aggresome depends on its phosphorylation status. a Validation of UPF1 hyperphosphorylation. HEK293T cells were transiently transfected with a plasmid expressing either Myc-UPF1-WT (wild type) or one of its variants. Two days later, the cells were harvested. Next, the extract of cells was treated with RNase A before IPs and was subjected to IPs using the anti (α)-Myc antibody. The samples before and after IPs were analyzed by western blotting; n = 2. Source data are provided as a Source Data File. b Immunostaining of CFTR-ΔF508 (green) and either Myc-UPF1-WT or its variants (red). HeLa cells stably expressing CFTR-ΔF508 were transiently transfected with the indicated plasmid expressing either Myc-UPF1-WT or one of its variants. Immunostaining images are representative of three independently processed biological replicates. Scale bar, 10 μm; n = 3.

    Article Snippet: Cell culture and chemical treatment HeLa and HEK293T cells (purchased from ATCC), and MEFs (a gift from Byung-Yoon Ahn, Korea University, Seoul, Korea) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Capricorn Scientific) containing 10% fetal bovine serum (Capricorn Scientific) and 1% penicillin/streptomycin (Capricorn Scientific).

    Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Immunostaining, Stable Transfection

    Measurements of different cell populations in spleen cells by flow cytometry using appropriate surface markers conjugated with different fluorochromes. Fluorescence signal of T-cell surface marker CD3e conjugated with PE-Vio770 (P1 = T cells) and B-cell surface marker CD19 conjugated with APC-Vio770 (P2 = B cells) on the cells from mice receiving (a) vehicle control or (b) lipopolysaccharide (LPS). The percentage of each cell population in mice (c) 24 h after LPS treatment (three doses, n = 4) or (d) 3 weeks after treatment with extracellular vesicles (10 doses, n = 10). Bars and error bars denote the mean and standard deviation, respectively, of experimental groups. PBS, phosphate-buffered saline; WT, wild-type HEK293T cells. * p

    Journal: Journal of Extracellular Vesicles

    Article Title: Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells

    doi: 10.1080/20013078.2017.1324730

    Figure Lengend Snippet: Measurements of different cell populations in spleen cells by flow cytometry using appropriate surface markers conjugated with different fluorochromes. Fluorescence signal of T-cell surface marker CD3e conjugated with PE-Vio770 (P1 = T cells) and B-cell surface marker CD19 conjugated with APC-Vio770 (P2 = B cells) on the cells from mice receiving (a) vehicle control or (b) lipopolysaccharide (LPS). The percentage of each cell population in mice (c) 24 h after LPS treatment (three doses, n = 4) or (d) 3 weeks after treatment with extracellular vesicles (10 doses, n = 10). Bars and error bars denote the mean and standard deviation, respectively, of experimental groups. PBS, phosphate-buffered saline; WT, wild-type HEK293T cells. * p

    Article Snippet: Cell lines Wild-type HEK293T cells (WT, ATCC CRL-11268) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, CA, USA) supplemented with 5% foetal bovine serum (Sigma, St Louis, MO, USA) that was depleted of EVs by ultracentrifugation (110,000 × g , 18 h).

    Techniques: Flow Cytometry, Cytometry, Fluorescence, Marker, Mouse Assay, Standard Deviation

    Analysis of Env incorporation into virions. HEK293T/17 cells were transfected with DNA encoding the HIV-1 virus. Three days after transfection the viral supernatants were pelleted by ultracentrifugation and separated by SDS-PAGE on a 4–12% bis-tris

    Journal: The Journal of Biological Chemistry

    Article Title: Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41 *

    doi: 10.1074/jbc.M113.529339

    Figure Lengend Snippet: Analysis of Env incorporation into virions. HEK293T/17 cells were transfected with DNA encoding the HIV-1 virus. Three days after transfection the viral supernatants were pelleted by ultracentrifugation and separated by SDS-PAGE on a 4–12% bis-tris

    Article Snippet: HEK293T/17 cells were transfected with pUC19–89.6 wild type or mutated DNA in a 6-well plate, and after 24 h the transfected cells were harvested using 1× citric saline for 4 min at 37 °C and diluted with 1 volume of 1× PBS.

    Techniques: Transfection, SDS Page

    Cell-to-cell fusion mediated by Env at the cell surface. A, cell-free supernatant from donor-transfected HEK293T/17 cells was added to the TZMbl target cells for 6 h in the presence of AZT. Luciferase activity is expressed as RLUs. Data represent one

    Journal: The Journal of Biological Chemistry

    Article Title: Unique Functional Properties of Conserved Arginine Residues in the Lentivirus Lytic Peptide Domains of the C-terminal Tail of HIV-1 gp41 *

    doi: 10.1074/jbc.M113.529339

    Figure Lengend Snippet: Cell-to-cell fusion mediated by Env at the cell surface. A, cell-free supernatant from donor-transfected HEK293T/17 cells was added to the TZMbl target cells for 6 h in the presence of AZT. Luciferase activity is expressed as RLUs. Data represent one

    Article Snippet: HEK293T/17 cells were transfected with pUC19–89.6 wild type or mutated DNA in a 6-well plate, and after 24 h the transfected cells were harvested using 1× citric saline for 4 min at 37 °C and diluted with 1 volume of 1× PBS.

    Techniques: Transfection, Luciferase, Activity Assay

    Evaluation of the effect of copy number on riboswitch dynamics. (A) Schematic representation of constructs generated through insertion of 1, 2, 3 or 4 copies of each riboswitch into the 3′-UTR and depiction of the mechanism of an ‘ON-type’ riboswitch in the presence or absence of activating ligand. hPGK; human phosphoglycerate kinase promoter, p(A); polyadenylation signal, SV40; Simian vacuolating virus 40 promoter, RS; riboswitch. Dual luciferase assay in HEK293T cells with (grey bars) or without (blue bars) the activating ligand at saturating levels for cells transfected with 1,2,3 or 4 copies of (B) K19, (C) L2Bulge9, (D) L2Bulge18tc, (E) GuaM8HDV or (F) TC45; n = 3 for all groups, green arrow represents the optimal copy number. Values are normalized to unmodified Firefly luciferase activity with standard error shown.

    Journal: Scientific Reports

    Article Title: Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD

    doi: 10.1038/s41598-018-29726-7

    Figure Lengend Snippet: Evaluation of the effect of copy number on riboswitch dynamics. (A) Schematic representation of constructs generated through insertion of 1, 2, 3 or 4 copies of each riboswitch into the 3′-UTR and depiction of the mechanism of an ‘ON-type’ riboswitch in the presence or absence of activating ligand. hPGK; human phosphoglycerate kinase promoter, p(A); polyadenylation signal, SV40; Simian vacuolating virus 40 promoter, RS; riboswitch. Dual luciferase assay in HEK293T cells with (grey bars) or without (blue bars) the activating ligand at saturating levels for cells transfected with 1,2,3 or 4 copies of (B) K19, (C) L2Bulge9, (D) L2Bulge18tc, (E) GuaM8HDV or (F) TC45; n = 3 for all groups, green arrow represents the optimal copy number. Values are normalized to unmodified Firefly luciferase activity with standard error shown.

    Article Snippet: Cell culture conditions HEK293T cells were obtained from ATCC (#CRL-11268, Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium with high glucose and GlutaMAX (Thermo Fisher Technologies, Carlsbad, CA).

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

    Assessment of riboswitch dosing kinetics in HEK293T cells. Normalized GFP fluorescence and representative GFP (inducible) and mCherry (non-inducible) images at varying concentrations of activating ligand for (A) K19, (B) L2Bulge9, (C) L2Bulge18tc, (D) GuaM8HDV and (E) TC45. Dynamic range is represented as fold-change. n = 3 , ** p > 0.01, *** p > 0.001, **** p > 0.001, One-Way ANOVA with Tukey’s post-hoc test. For each group, respective fluorescent channels were recorded with constant exposure and gain; scale bars = 50 μM.

    Journal: Scientific Reports

    Article Title: Development of an inducible anti-VEGF rAAV gene therapy strategy for the treatment of wet AMD

    doi: 10.1038/s41598-018-29726-7

    Figure Lengend Snippet: Assessment of riboswitch dosing kinetics in HEK293T cells. Normalized GFP fluorescence and representative GFP (inducible) and mCherry (non-inducible) images at varying concentrations of activating ligand for (A) K19, (B) L2Bulge9, (C) L2Bulge18tc, (D) GuaM8HDV and (E) TC45. Dynamic range is represented as fold-change. n = 3 , ** p > 0.01, *** p > 0.001, **** p > 0.001, One-Way ANOVA with Tukey’s post-hoc test. For each group, respective fluorescent channels were recorded with constant exposure and gain; scale bars = 50 μM.

    Article Snippet: Cell culture conditions HEK293T cells were obtained from ATCC (#CRL-11268, Manassas, VA) and cultured in Dulbecco’s Modified Eagle Medium with high glucose and GlutaMAX (Thermo Fisher Technologies, Carlsbad, CA).

    Techniques: Fluorescence

    Optimization of linear-DNA based transfection. (A) Optimization of the amount of linear-DNA based transfection. 0.05 , 0.1, 0.2 linear and 0.2 μg of plasmid DNA encoding AtCRY2 were transfected with the optimized Lipofectamine 2000, respectively. After 24-h incubation, total protein of each sample was probed by the antibody to AtCRY2, stripped and re-probed by the antibody to Actin. (B) Efficiency testing of different transfection reagents. 0.2 μl linear DNA encoding AtCRY2 were transfected with Calcium Phosphate, Lipofectamine ® 2000 or Lipofectamine ® 3000, the optimized protocols were applied for those transfection reagents, respectively. After transfection and incubation, the immunoblot was performed as described in (A) . (C) Efficiency testing of co-transfection with different transfection reagents. 0.3 μg linear DNA encoding AtCRY2 and Myc-AtCIB1 were co-transfected with different transfection reagent. The expression level of AtCRY2 and Myc-AtCIB1, transfected with indicated transfection reagent, was shown. The immunoblot was performed as described in (A) . (D) The microscopy of HEK293T cells transfected with AtCRY2–GFP. Cells were transfected and then incubated with indicated time before microscopy. The GFP and BF (Bright field) channels were merged with Zeiss Zen2 software. (E) The efficiency testing of different post-transfection incubation time. After transfection and incubation for indicated time, cells were detached by Recombinant trypsin and re-suspended with PBS (pH 7.2). The efficiency of AtCRY2–GFP was calculated with the following equation: [cell with GFP signal/total counted cell] × 100%.

    Journal: Frontiers in Plant Science

    Article Title: Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    doi: 10.3389/fpls.2016.00940

    Figure Lengend Snippet: Optimization of linear-DNA based transfection. (A) Optimization of the amount of linear-DNA based transfection. 0.05 , 0.1, 0.2 linear and 0.2 μg of plasmid DNA encoding AtCRY2 were transfected with the optimized Lipofectamine 2000, respectively. After 24-h incubation, total protein of each sample was probed by the antibody to AtCRY2, stripped and re-probed by the antibody to Actin. (B) Efficiency testing of different transfection reagents. 0.2 μl linear DNA encoding AtCRY2 were transfected with Calcium Phosphate, Lipofectamine ® 2000 or Lipofectamine ® 3000, the optimized protocols were applied for those transfection reagents, respectively. After transfection and incubation, the immunoblot was performed as described in (A) . (C) Efficiency testing of co-transfection with different transfection reagents. 0.3 μg linear DNA encoding AtCRY2 and Myc-AtCIB1 were co-transfected with different transfection reagent. The expression level of AtCRY2 and Myc-AtCIB1, transfected with indicated transfection reagent, was shown. The immunoblot was performed as described in (A) . (D) The microscopy of HEK293T cells transfected with AtCRY2–GFP. Cells were transfected and then incubated with indicated time before microscopy. The GFP and BF (Bright field) channels were merged with Zeiss Zen2 software. (E) The efficiency testing of different post-transfection incubation time. After transfection and incubation for indicated time, cells were detached by Recombinant trypsin and re-suspended with PBS (pH 7.2). The efficiency of AtCRY2–GFP was calculated with the following equation: [cell with GFP signal/total counted cell] × 100%.

    Article Snippet: HEK293T Cell Culture HEK293T (ATCC® CRL-11268TM ) cells were maintained in DMEM (Thermo, 10569-044) supplemented with 10% (v/v) FBS (Thermo, 10100147), 100 U/ml penicillin, and 100 mg/ml streptomycin (Hyclone, SV30010) in Cell Culture Flask, T75 (Eppendorf, 0030711.122).

    Techniques: Transfection, Plasmid Preparation, Incubation, Cotransfection, Expressing, Microscopy, Software, Recombinant

    Workflow of PCR product based HEK293 cell transfection (96-well-plate format with Lipofectamine 2000). (1) HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin, and 100 mg/ml streptomycin under the condition of 37°C, 5% CO 2 . Cells were seeded into 96-well-plate for ∼24 h before transfection. At the same day, SV40 promoter and plant ORF were amplified, respectively, with primers that include 15 bp of overlap sequence. The linear DNA including SV40 promoter and plant ORF were then generated with overlap PCR, purified and quantified for transfection. (2) HEK293T cells were cultured until 80–90% confluence is reached. Added 10 μl Opti-MEM into a sterile tube. (3) Added 0.2 μg linear DNA and vortex. (4) Added 0.6 μl Lipofectamine 2000 and vortex. (5) Incubated at room temperature for 5 min. (6) Added the mixture to 96-well-plate, 10 μl per well, and shook the plate gently. (7) After incubation for 16–32 h, cells were then collected and lysed for Biochemistry assay.

    Journal: Frontiers in Plant Science

    Article Title: Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    doi: 10.3389/fpls.2016.00940

    Figure Lengend Snippet: Workflow of PCR product based HEK293 cell transfection (96-well-plate format with Lipofectamine 2000). (1) HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/ml penicillin, and 100 mg/ml streptomycin under the condition of 37°C, 5% CO 2 . Cells were seeded into 96-well-plate for ∼24 h before transfection. At the same day, SV40 promoter and plant ORF were amplified, respectively, with primers that include 15 bp of overlap sequence. The linear DNA including SV40 promoter and plant ORF were then generated with overlap PCR, purified and quantified for transfection. (2) HEK293T cells were cultured until 80–90% confluence is reached. Added 10 μl Opti-MEM into a sterile tube. (3) Added 0.2 μg linear DNA and vortex. (4) Added 0.6 μl Lipofectamine 2000 and vortex. (5) Incubated at room temperature for 5 min. (6) Added the mixture to 96-well-plate, 10 μl per well, and shook the plate gently. (7) After incubation for 16–32 h, cells were then collected and lysed for Biochemistry assay.

    Article Snippet: HEK293T Cell Culture HEK293T (ATCC® CRL-11268TM ) cells were maintained in DMEM (Thermo, 10569-044) supplemented with 10% (v/v) FBS (Thermo, 10100147), 100 U/ml penicillin, and 100 mg/ml streptomycin (Hyclone, SV30010) in Cell Culture Flask, T75 (Eppendorf, 0030711.122).

    Techniques: Polymerase Chain Reaction, Transfection, Cell Culture, Amplification, Sequencing, Generated, Purification, Incubation

    Biochemical analysis of Arabidopsis CRY2 expressed in HEK293T cell. (A) For expression and purification of the recombinant protein, 6xHis-AtCRY2 and 6xHis-AtCRY2 D387A were transfected with Calcium Phosphate, respectively. After 24 hours of incubation, cells were collected and lysed. Recombinant AtCRY2 and AtCRY2 D387A were purified with Ni-NTA agarose beads. The AtCRY2 exhibited a faint yellow color after enrichment. (B) The absorption spectrum of recombinant AtCRY2 and AtCRY2 D387A were detected by a full-length spectrophotometer, and the related absorption curve was calculated by the following equation: [absorption of CRY2 - absorption of CRY2 D387A ]. (C,D) Co-IP analysis showing blue light dependent interaction of AtCRY2–AtCIB1 (C) and AtCRY2–AtSPA1 (D) in mammalian cells. The HEK293T cells co-transfected with AtCRY2–AtCIB1 or AtCRY2–AtSPA1 were incubated in dark condition. Cells were irradiated with blue light (50 μmol m -2 s -1 ) for indicated time before harvest. Total protein extraction and immunoprecipitation (IP) product prepared by AtCRY2 antibody were first probed with AtCRY2 antibody, stripped, and re-probed with Myc antibody.

    Journal: Frontiers in Plant Science

    Article Title: Using HEK293T Expression System to Study Photoactive Plant Cryptochromes

    doi: 10.3389/fpls.2016.00940

    Figure Lengend Snippet: Biochemical analysis of Arabidopsis CRY2 expressed in HEK293T cell. (A) For expression and purification of the recombinant protein, 6xHis-AtCRY2 and 6xHis-AtCRY2 D387A were transfected with Calcium Phosphate, respectively. After 24 hours of incubation, cells were collected and lysed. Recombinant AtCRY2 and AtCRY2 D387A were purified with Ni-NTA agarose beads. The AtCRY2 exhibited a faint yellow color after enrichment. (B) The absorption spectrum of recombinant AtCRY2 and AtCRY2 D387A were detected by a full-length spectrophotometer, and the related absorption curve was calculated by the following equation: [absorption of CRY2 - absorption of CRY2 D387A ]. (C,D) Co-IP analysis showing blue light dependent interaction of AtCRY2–AtCIB1 (C) and AtCRY2–AtSPA1 (D) in mammalian cells. The HEK293T cells co-transfected with AtCRY2–AtCIB1 or AtCRY2–AtSPA1 were incubated in dark condition. Cells were irradiated with blue light (50 μmol m -2 s -1 ) for indicated time before harvest. Total protein extraction and immunoprecipitation (IP) product prepared by AtCRY2 antibody were first probed with AtCRY2 antibody, stripped, and re-probed with Myc antibody.

    Article Snippet: HEK293T Cell Culture HEK293T (ATCC® CRL-11268TM ) cells were maintained in DMEM (Thermo, 10569-044) supplemented with 10% (v/v) FBS (Thermo, 10100147), 100 U/ml penicillin, and 100 mg/ml streptomycin (Hyclone, SV30010) in Cell Culture Flask, T75 (Eppendorf, 0030711.122).

    Techniques: Expressing, Purification, Recombinant, Transfection, Incubation, Spectrophotometry, Co-Immunoprecipitation Assay, Irradiation, Protein Extraction, Immunoprecipitation

    FIN mediates the COX2/PGE 2 pathway by increasing the activity of COX2 promoter. The plasmid harboring a COX2 promoter fragment and a firefly luciferase (FL) gene (5ng/well separately) was transfected into HEK293T cells in a 24-well-plate and a CMV-Renilla luciferase (RL) plasmid (10ng/well) served as a transfection control. FIN (50μM) was treated for 6 h before harvesting the cells for dual-luciferase assay. Transfected cells treated with DMSO were set as negative control. Results are shown in mean ± SEM and representative of 3 independent experiments. **p

    Journal: PLoS ONE

    Article Title: Finasteride Enhances the Generation of Human Myeloid-Derived Suppressor Cells by Up-Regulating the COX2/PGE2 Pathway

    doi: 10.1371/journal.pone.0156549

    Figure Lengend Snippet: FIN mediates the COX2/PGE 2 pathway by increasing the activity of COX2 promoter. The plasmid harboring a COX2 promoter fragment and a firefly luciferase (FL) gene (5ng/well separately) was transfected into HEK293T cells in a 24-well-plate and a CMV-Renilla luciferase (RL) plasmid (10ng/well) served as a transfection control. FIN (50μM) was treated for 6 h before harvesting the cells for dual-luciferase assay. Transfected cells treated with DMSO were set as negative control. Results are shown in mean ± SEM and representative of 3 independent experiments. **p

    Article Snippet: Cell culture HEK293T cells (Cat. No. CRL-1573)were purchased from ATCC (American Type Culture Collection) and maintained in the conditioned Dulbecco’s Modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA) with 10% fetal calf serum (Gibco), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco) at 37°C and 5% CO2.

    Techniques: Activity Assay, Plasmid Preparation, Luciferase, Transfection, Negative Control

    IL-1β induces the expression of miR-31, which affects E-selectin abundance a. The monolayer of HUVEC endothelial cells was treated with IL-1β (20ng/ml) or TNFα (10ng/ml) for indicated hours. Western blotting monitored the expression of E-selectin and Actin used as loading control. b. The miR-31 level was measured by quantitative reverse transcription-PCR and the snRNA U6 was used as the normalization control. c. Endothelial cells were transfected with 30nM of either anti-miR-31 (+) or corresponding inhibitor negative control (−) before the addition of IL-1β. The Western blots shown are representative of three independent experiments. The endogenous GAPDH was used as loading control. d. Top: The representation of miR-31 base-pairing with either E-selectin wild-type (wt) or mutated (mut) 3′ UTR sequence. The nucleotides in the gray box represent the seed region of miR-31, region important for target interaction. Bottom: E-selectin 3′UTR mediated reporter assays. Vector expressing luciferase reporter under the regulation of either wild-type (black bars) or mutated (white bars) E-selectin 3′UTR were transfected into HEK293T cells. 48 hours after transfection, relative luciferase activities were measured. Another set of reporters transfected cells were also transfected with 30nM of either anti-miR-31 or corresponding inhibitor negative control (ctl) to further test the contribution of miR-31 in E-selectin regulation. The error bars shown in panel b and d represent standard errors of three and six independent experiments, respectively. The significance was analyzed using a Student's t -test (* p

    Journal: Oncotarget

    Article Title: p38 and JNK pathways control E-selectin-dependent extravasation of colon cancer cells by modulating miR-31 transcription

    doi: 10.18632/oncotarget.13779

    Figure Lengend Snippet: IL-1β induces the expression of miR-31, which affects E-selectin abundance a. The monolayer of HUVEC endothelial cells was treated with IL-1β (20ng/ml) or TNFα (10ng/ml) for indicated hours. Western blotting monitored the expression of E-selectin and Actin used as loading control. b. The miR-31 level was measured by quantitative reverse transcription-PCR and the snRNA U6 was used as the normalization control. c. Endothelial cells were transfected with 30nM of either anti-miR-31 (+) or corresponding inhibitor negative control (−) before the addition of IL-1β. The Western blots shown are representative of three independent experiments. The endogenous GAPDH was used as loading control. d. Top: The representation of miR-31 base-pairing with either E-selectin wild-type (wt) or mutated (mut) 3′ UTR sequence. The nucleotides in the gray box represent the seed region of miR-31, region important for target interaction. Bottom: E-selectin 3′UTR mediated reporter assays. Vector expressing luciferase reporter under the regulation of either wild-type (black bars) or mutated (white bars) E-selectin 3′UTR were transfected into HEK293T cells. 48 hours after transfection, relative luciferase activities were measured. Another set of reporters transfected cells were also transfected with 30nM of either anti-miR-31 or corresponding inhibitor negative control (ctl) to further test the contribution of miR-31 in E-selectin regulation. The error bars shown in panel b and d represent standard errors of three and six independent experiments, respectively. The significance was analyzed using a Student's t -test (* p

    Article Snippet: HEK293T human embryonic kidney cells (ATCC) were cultivated in DMEM (Lonza, Allendale, NJ) supplemented with 10% FBS.

    Techniques: Expressing, Western Blot, Polymerase Chain Reaction, Transfection, Negative Control, Sequencing, Plasmid Preparation, Luciferase, CTL Assay

    CYP26A1 gene expression is sensitive to RA treatment in HNF4α-containing HepG2 cells. HepG2 or HEK293T cells were grown in 6-well plates and treated with either ethanol as the vehicle control or RA in triplicate for 4 h and then collected for

    Journal: Journal of cellular biochemistry

    Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) in Coordination With Retinoic Acid Receptors Increases all-trans-Retinoic Acid-Dependent CYP26A1 Gene Expression in HepG2 Human Hepatocytes

    doi: 10.1002/jcb.24839

    Figure Lengend Snippet: CYP26A1 gene expression is sensitive to RA treatment in HNF4α-containing HepG2 cells. HepG2 or HEK293T cells were grown in 6-well plates and treated with either ethanol as the vehicle control or RA in triplicate for 4 h and then collected for

    Article Snippet: HepG2 cells, and human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin at 37 °C in a 5% CO2 -air incubator.

    Techniques: Expressing

    HNF4α enhances CYP26A1 promoter activity in HepG2 cells but not in HEK293T cells treated with RA. Cells grown in 24-well plates were cotransfected in triplicate with p GL3-Basic- luc -hCYP26A1 promoter containing full-length human CYP26A1 promoter

    Journal: Journal of cellular biochemistry

    Article Title: Hepatocyte Nuclear Factor 4α (HNF4α) in Coordination With Retinoic Acid Receptors Increases all-trans-Retinoic Acid-Dependent CYP26A1 Gene Expression in HepG2 Human Hepatocytes

    doi: 10.1002/jcb.24839

    Figure Lengend Snippet: HNF4α enhances CYP26A1 promoter activity in HepG2 cells but not in HEK293T cells treated with RA. Cells grown in 24-well plates were cotransfected in triplicate with p GL3-Basic- luc -hCYP26A1 promoter containing full-length human CYP26A1 promoter

    Article Snippet: HepG2 cells, and human embryonic kidney HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and 0.5% penicillin-streptomycin at 37 °C in a 5% CO2 -air incubator.

    Techniques: Activity Assay

    Epilepsy‐associated mutations alter the function of the Nav1.2 channel. (a) Representative whole‐cell patch‐clamp traces of voltage‐dependent currents recorded from HEK293T cells transfected with either Nav1.2 wild‐type or mutant channels. Cells were held at −120 mV and sodium currents were evoked by a series of depolarizing pulses (100 ms) to potentials ranging from −80 mV to +90 mV in steps of 5 mV (inset). (b) Normalized I‒V curves of peak sodium current density (in pA/pF) versus voltage for WT and mutations. (c) Voltage dependence of activation obtained by plotting the normalized conductance against test potentials with equation G/G max = 1/(1 + exp(V 0.5 − V)/k, where the G max is the maximum conductance, V 0.5 is the half‐maximal activation potential and k is the slope factor. the curve is fitted to a Boltzmann function. (d) Steady state of fast inactivation of WT and mutant Nav1.2 channels. The voltage dependence of fast inactivation was assessed by applying a double‐pulse protocol: 500‐ms prepulses were applied from −150 to 0 mV in steps of 10 mV and followed by a test pulse to −10 mV (inset). The steady‐state fast inactivation curve was fitted by the Boltzmann equation (I/Imax = {1 + exp[(V − V 0.5 )/k]} − 1). (e) Window currents of WT the mutations. Activation curves (fraction of maximum conductance, G/G max ) and steady‐state inactivation (fraction of maximum current, I/Imax) of WT and the mutations are enlarged and plotted together to show the window currents. (f) The time course of recovery from fast inactivation. Recovery from fast inactivation was assessed by a two‐pulse recovery protocol with varying time intervals between a 500‐ms inactivating prepulse and a test pulse to −10 mV (inset). The time course of recovery from inactivation was fitted with a double‐exponential function to generate τ1 and τ2. All fitting results are listed in Table 1 . Data are presented as the mean ± SEM

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: Electrophysiological features: The next precise step for SCN2A developmental epileptic encephalopathy, et al. Electrophysiological features: The next precise step for SCN2A developmental epileptic encephalopathy

    doi: 10.1002/mgg3.1250

    Figure Lengend Snippet: Epilepsy‐associated mutations alter the function of the Nav1.2 channel. (a) Representative whole‐cell patch‐clamp traces of voltage‐dependent currents recorded from HEK293T cells transfected with either Nav1.2 wild‐type or mutant channels. Cells were held at −120 mV and sodium currents were evoked by a series of depolarizing pulses (100 ms) to potentials ranging from −80 mV to +90 mV in steps of 5 mV (inset). (b) Normalized I‒V curves of peak sodium current density (in pA/pF) versus voltage for WT and mutations. (c) Voltage dependence of activation obtained by plotting the normalized conductance against test potentials with equation G/G max = 1/(1 + exp(V 0.5 − V)/k, where the G max is the maximum conductance, V 0.5 is the half‐maximal activation potential and k is the slope factor. the curve is fitted to a Boltzmann function. (d) Steady state of fast inactivation of WT and mutant Nav1.2 channels. The voltage dependence of fast inactivation was assessed by applying a double‐pulse protocol: 500‐ms prepulses were applied from −150 to 0 mV in steps of 10 mV and followed by a test pulse to −10 mV (inset). The steady‐state fast inactivation curve was fitted by the Boltzmann equation (I/Imax = {1 + exp[(V − V 0.5 )/k]} − 1). (e) Window currents of WT the mutations. Activation curves (fraction of maximum conductance, G/G max ) and steady‐state inactivation (fraction of maximum current, I/Imax) of WT and the mutations are enlarged and plotted together to show the window currents. (f) The time course of recovery from fast inactivation. Recovery from fast inactivation was assessed by a two‐pulse recovery protocol with varying time intervals between a 500‐ms inactivating prepulse and a test pulse to −10 mV (inset). The time course of recovery from inactivation was fitted with a double‐exponential function to generate τ1 and τ2. All fitting results are listed in Table 1 . Data are presented as the mean ± SEM

    Article Snippet: 2.4 Cell culture and transfection HEK293T cells were obtained from ATCC and maintained at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco).

    Techniques: Patch Clamp, Transfection, Mutagenesis, Activation Assay