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    Structured Review

    ATCC hek293 cells
    Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 <t>HEK293</t> cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.
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    1) Product Images from "Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity"

    Article Title: Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

    Journal: Nature chemical biology

    doi: 10.1038/s41589-021-00878-4

    Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.
    Figure Legend Snippet: Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.

    Techniques Used: Mass Spectrometry, CRISPR, Serial Dilution, Concentration Assay, Expressing, Two Tailed Test

    SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL. a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC 50 values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC 50 values for each compound are shown for both permeabilized and live ce lls. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.
    Figure Legend Snippet: SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL. a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC 50 values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC 50 values for each compound are shown for both permeabilized and live ce lls. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.

    Techniques Used: Size-exclusion Chromatography, Incubation, Mutagenesis, Biosensor Assay, Transfection, Serial Dilution, Concentration Assay, Binding Assay, Titration, Expressing, Variant Assay

    Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader. a,b) Inspection of ternary complex crystal structures of VHL:MZ1:BRD4 BD2 (a, PDB:5T35) and BRD4 BD2 :MT1:BRD4 BD2 (b, PDB 5JWM) guided the identification of solvent-exposed region for chemical branching of linkers in trivalent PROTAC design. Chemical structures of parent bivalent molecules MZ1 and MT1 are shown. c) Chemical structures of designed trivalent PROTACs SIM1-6 based on VHL and CRBN E3 ligase ligands. d) Immunoblot analysis of BRD2, BRD3, BRD4 after treatment of HEK293 cells with 1μM PROTACs or DMSO for 4h, performed as n=1. Full blots are supplied as Source Data Fig. 1 . e) Protein levels of BRD2, BRD3, BRD4 in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 for 4h. Quantification of BET protein levels was done relative to DMSO control and shown plots used to measure the tabulated DC 50 values. Corresponding blots are in Extended Data Fig. 1c , and full blots are supplied as Source Data Fig. 1 . f) Cell viability of MV4;11 AML cell line following treatment with PROTACs or DMSO for 48h in three replicates for each concentration point. g) Chemical structures of SIM1 and its designed negative controls, ( R,S )-SIM1 and cis-SIM1. Reversed stereocenters are indicated by asterisks. h) Immunoblot of degradation of BET proteins in HEK293 cells after treatment with indicated compounds at 1μM or DMSO for 4h. Full blots are supplied as Source Data Fig. 1 . i) CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells were treated with 100nM of DMSO, MZ1, ( R,S )-SIM1, and both 10nM and 100nM of SIM1 in replicate plates for washout experiments. Media containing the 10nM and/or 100nM compounds was removed at 3.5h, indicated on the graphs, and replaced with media lacking compounds for the remainder of the experiment. Luminescence (RLU) was continuously monitored over a 50h time period and is plotted normalized to the DMSO control as Fractional RLU.
    Figure Legend Snippet: Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader. a,b) Inspection of ternary complex crystal structures of VHL:MZ1:BRD4 BD2 (a, PDB:5T35) and BRD4 BD2 :MT1:BRD4 BD2 (b, PDB 5JWM) guided the identification of solvent-exposed region for chemical branching of linkers in trivalent PROTAC design. Chemical structures of parent bivalent molecules MZ1 and MT1 are shown. c) Chemical structures of designed trivalent PROTACs SIM1-6 based on VHL and CRBN E3 ligase ligands. d) Immunoblot analysis of BRD2, BRD3, BRD4 after treatment of HEK293 cells with 1μM PROTACs or DMSO for 4h, performed as n=1. Full blots are supplied as Source Data Fig. 1 . e) Protein levels of BRD2, BRD3, BRD4 in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 for 4h. Quantification of BET protein levels was done relative to DMSO control and shown plots used to measure the tabulated DC 50 values. Corresponding blots are in Extended Data Fig. 1c , and full blots are supplied as Source Data Fig. 1 . f) Cell viability of MV4;11 AML cell line following treatment with PROTACs or DMSO for 48h in three replicates for each concentration point. g) Chemical structures of SIM1 and its designed negative controls, ( R,S )-SIM1 and cis-SIM1. Reversed stereocenters are indicated by asterisks. h) Immunoblot of degradation of BET proteins in HEK293 cells after treatment with indicated compounds at 1μM or DMSO for 4h. Full blots are supplied as Source Data Fig. 1 . i) CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells were treated with 100nM of DMSO, MZ1, ( R,S )-SIM1, and both 10nM and 100nM of SIM1 in replicate plates for washout experiments. Media containing the 10nM and/or 100nM compounds was removed at 3.5h, indicated on the graphs, and replaced with media lacking compounds for the remainder of the experiment. Luminescence (RLU) was continuously monitored over a 50h time period and is plotted normalized to the DMSO control as Fractional RLU.

    Techniques Used: Concentration Assay, CRISPR

    2) Product Images from "EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation"

    Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

    Journal: BMC Pulmonary Medicine

    doi: 10.1186/s12890-021-01553-z

    Effect of oncogenic EML4-ALK tyrosine kinase on the biological behaviors of lung cancer cells. a EML4-ALK levels in transfected H2228 and HEK293 cells. b Increased apoptosis in H2228 cells following EML4-ALK knockdown measured by flow cytometry. c Cell viability, measured by the CCK8 assay, was increased by EML4-ALK expression in HEK293 cells. d – e Viability and proliferation of H2228 cells decreased after knockdown of EML4-ALK or STAT6. f H2228 cell viability following treatment with ALK or JAK-STAT pathway inhibitors as measured by the CCK-8 assay. * P
    Figure Legend Snippet: Effect of oncogenic EML4-ALK tyrosine kinase on the biological behaviors of lung cancer cells. a EML4-ALK levels in transfected H2228 and HEK293 cells. b Increased apoptosis in H2228 cells following EML4-ALK knockdown measured by flow cytometry. c Cell viability, measured by the CCK8 assay, was increased by EML4-ALK expression in HEK293 cells. d – e Viability and proliferation of H2228 cells decreased after knockdown of EML4-ALK or STAT6. f H2228 cell viability following treatment with ALK or JAK-STAT pathway inhibitors as measured by the CCK-8 assay. * P

    Techniques Used: Transfection, Flow Cytometry, CCK-8 Assay, Expressing

    Microarray analysis of H2228 cells without or with EML4-ALK knockdown and of HEK293 cells expressing or not the EML4-ALK variant 3. a The heat map shows the regulation of a number of JAK2-STAT pathway genes. b Expression of JAK2-STAT pathway-related genes measured by real-time PCR
    Figure Legend Snippet: Microarray analysis of H2228 cells without or with EML4-ALK knockdown and of HEK293 cells expressing or not the EML4-ALK variant 3. a The heat map shows the regulation of a number of JAK2-STAT pathway genes. b Expression of JAK2-STAT pathway-related genes measured by real-time PCR

    Techniques Used: Microarray, Expressing, Variant Assay, Real-time Polymerase Chain Reaction

    Interactions between components of the JAK2-STAT pathway and EML4-ALK. a Active STAT6 and JAK2 colocalized with ALK in EML4-ALK-transfected HEK293 cells and in H2228 cells. b : ALK and phosphorylated STAT6 protein were co-immunoprecipitated with JAK2 in EML4-ALK-transfected HEK293 cells
    Figure Legend Snippet: Interactions between components of the JAK2-STAT pathway and EML4-ALK. a Active STAT6 and JAK2 colocalized with ALK in EML4-ALK-transfected HEK293 cells and in H2228 cells. b : ALK and phosphorylated STAT6 protein were co-immunoprecipitated with JAK2 in EML4-ALK-transfected HEK293 cells

    Techniques Used: Transfection, Immunoprecipitation

    EML4-ALK transfection activated JAK2, STAT1, STAT3, STAT5, and STAT6 in HEK293 and NIH3T3 cells
    Figure Legend Snippet: EML4-ALK transfection activated JAK2, STAT1, STAT3, STAT5, and STAT6 in HEK293 and NIH3T3 cells

    Techniques Used: Transfection

    3) Product Images from "Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon"

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    Journal: Journal of Virology

    doi: 10.1128/JVI.02095-17

    Overexpression of EGFR facilitates PEDV infection. (A and C) IPEC-J2 (A) and HEK293 (C) cells were transfected with pAAV/EGFR and pAAV vector control (con) for 24 h. The cells were then infected with PEDV for 48 h or 24 h. Detergent lysates were subjected to immunoblotting with the indicated antibodies. (B and D) EGFR overexpression enhances the levels of PEDV RNA. Total RNA was extracted from IPEC-J2 cells at 48 h postinfection (hpi) (B) or from HEK293 cells at 24 hpi (D) and analyzed by quantitative RT-PCR. (E and F) EGFR overexpression promotes PEDV titers. Virus samples were collected from IPEC-J2 cells at 48 hpi (E) or from HEK293 cells at 24 hpi (F) and measured by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P
    Figure Legend Snippet: Overexpression of EGFR facilitates PEDV infection. (A and C) IPEC-J2 (A) and HEK293 (C) cells were transfected with pAAV/EGFR and pAAV vector control (con) for 24 h. The cells were then infected with PEDV for 48 h or 24 h. Detergent lysates were subjected to immunoblotting with the indicated antibodies. (B and D) EGFR overexpression enhances the levels of PEDV RNA. Total RNA was extracted from IPEC-J2 cells at 48 h postinfection (hpi) (B) or from HEK293 cells at 24 hpi (D) and analyzed by quantitative RT-PCR. (E and F) EGFR overexpression promotes PEDV titers. Virus samples were collected from IPEC-J2 cells at 48 hpi (E) or from HEK293 cells at 24 hpi (F) and measured by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Techniques Used: Over Expression, Infection, Transfection, Plasmid Preparation, Quantitative RT-PCR

    STAT3 is involved in PEDV infection. (A and B) Reduction of virus loads by S3I-201 is concentration dependent. IPEC-J2 (A) and HEK293 (B) cells were treated with different concentrations of the STAT3 inhibitor S3I-201 for 24 h, followed by infection with PEDV. At 48 hpi (IPEC-J2) or 24 hpi (HEK293), virus infection was determined by Western blotting with antibodies to PEDV N protein and β-actin (loading control). Densitometric data for the PEDV N protein/actin ratio from three independent experiments are expressed as means and SD. Virus RNA levels were also assessed by quantitative RT-PCR. (C and D) Knockdown of STAT3 expression reduced PEDV infection. Cells were transfected with STAT3-specific siRNA for 24 h and then infected with PEDV for 48 h or 24 h. STAT3 knockdown efficiency was verified by quantitative RT-PCR and Western blotting. The effect of STAT3 knockdown on virus loads was determined by Western blotting and quantitative RT-PCR. (E and F) Overexpression of STAT3 increased PEDV infection. IPEC-J2 (E) and HEK293 (F) cells were transfected with pAAV/STAT3 or vector control. Twenty-four hours later, the cells were incubated with PEDV for an additional 48 h or 24 h. The levels of STAT3 protein and PEDV N protein were analyzed by Western blotting. Total RNA was also extracted from the cells, and the viral RNA levels were then assessed by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P
    Figure Legend Snippet: STAT3 is involved in PEDV infection. (A and B) Reduction of virus loads by S3I-201 is concentration dependent. IPEC-J2 (A) and HEK293 (B) cells were treated with different concentrations of the STAT3 inhibitor S3I-201 for 24 h, followed by infection with PEDV. At 48 hpi (IPEC-J2) or 24 hpi (HEK293), virus infection was determined by Western blotting with antibodies to PEDV N protein and β-actin (loading control). Densitometric data for the PEDV N protein/actin ratio from three independent experiments are expressed as means and SD. Virus RNA levels were also assessed by quantitative RT-PCR. (C and D) Knockdown of STAT3 expression reduced PEDV infection. Cells were transfected with STAT3-specific siRNA for 24 h and then infected with PEDV for 48 h or 24 h. STAT3 knockdown efficiency was verified by quantitative RT-PCR and Western blotting. The effect of STAT3 knockdown on virus loads was determined by Western blotting and quantitative RT-PCR. (E and F) Overexpression of STAT3 increased PEDV infection. IPEC-J2 (E) and HEK293 (F) cells were transfected with pAAV/STAT3 or vector control. Twenty-four hours later, the cells were incubated with PEDV for an additional 48 h or 24 h. The levels of STAT3 protein and PEDV N protein were analyzed by Western blotting. Total RNA was also extracted from the cells, and the viral RNA levels were then assessed by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P

    Techniques Used: Infection, Concentration Assay, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Over Expression, Plasmid Preparation, Incubation

    Depletion of EGFR expression enhanced the levels of several ISGs. IPEC-J2 (A) and HEK293 (B) cells were transfected with control siRNA or EGFR-specific siRNA for 24 h. Total RNA was extracted, and the mRNA levels of MxA , ISG15 , and IFN-β were determined by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P
    Figure Legend Snippet: Depletion of EGFR expression enhanced the levels of several ISGs. IPEC-J2 (A) and HEK293 (B) cells were transfected with control siRNA or EGFR-specific siRNA for 24 h. Total RNA was extracted, and the mRNA levels of MxA , ISG15 , and IFN-β were determined by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR

    EGFR inhibitors reduce PEDV infection. (A) Effects of inhibitors on EGFR function. IPEC-J2 and HEK293 cells were treated with the carrier control DMSO or EGFR-specific inhibitors, erlotinib (Erl) and gefitinib (Gef), at different concentrations for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. IPEC-J2 and HEK293 cells were also pretreated with an EGFR-specific inhibitor, Erl or Gef, at 10 μM or with DMSO for 12 h, followed by EGF stimulation (10 ng/ml) for 15 min. The levels of pEGFR and β-actin were analyzed by Western blotting. (B) EGFR inhibitors decrease the number of PEDV-positive cells. IPEC-J2 and HEK293 cells were pretreated with DMSO or EGFR inhibitors, Erl and Gef, at 10 μM for 12 h. After washing, the cells were infected with PEDV or mock control in the absence or presence of inhibitors. At 48 hpi or 24 hpi, the cell monolayers were fixed and examined for PEDV infection by IFA with an anti-PEDV spike protein MAb (3F12). The number of PEDV-positive cells was calculated. (C) Reduction of PEDV N protein by EGFR inhibitors is concentration dependent. Detergent lysates collected from IPEC-J2 and HEK293 cells were subjected to immunoblotting with antibodies as indicated. (D) EGFR inhibitors decreased PEDV RNA levels, as determined by quantitative RT-PCR. (E) Virus titers were reduced after inhibitor treatment, as detected by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P
    Figure Legend Snippet: EGFR inhibitors reduce PEDV infection. (A) Effects of inhibitors on EGFR function. IPEC-J2 and HEK293 cells were treated with the carrier control DMSO or EGFR-specific inhibitors, erlotinib (Erl) and gefitinib (Gef), at different concentrations for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. IPEC-J2 and HEK293 cells were also pretreated with an EGFR-specific inhibitor, Erl or Gef, at 10 μM or with DMSO for 12 h, followed by EGF stimulation (10 ng/ml) for 15 min. The levels of pEGFR and β-actin were analyzed by Western blotting. (B) EGFR inhibitors decrease the number of PEDV-positive cells. IPEC-J2 and HEK293 cells were pretreated with DMSO or EGFR inhibitors, Erl and Gef, at 10 μM for 12 h. After washing, the cells were infected with PEDV or mock control in the absence or presence of inhibitors. At 48 hpi or 24 hpi, the cell monolayers were fixed and examined for PEDV infection by IFA with an anti-PEDV spike protein MAb (3F12). The number of PEDV-positive cells was calculated. (C) Reduction of PEDV N protein by EGFR inhibitors is concentration dependent. Detergent lysates collected from IPEC-J2 and HEK293 cells were subjected to immunoblotting with antibodies as indicated. (D) EGFR inhibitors decreased PEDV RNA levels, as determined by quantitative RT-PCR. (E) Virus titers were reduced after inhibitor treatment, as detected by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Techniques Used: Infection, CCK-8 Assay, Western Blot, Immunofluorescence, Concentration Assay, Quantitative RT-PCR

    (A to C) PEDV infection induces EGFR phosphorylation. IPEC-J2, HEK293, and Vero E6 cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed with PBS, and the cells were then cultured at 37°C for different times as indicated. Mock-infected cells were used as a control. Detergent lysates collected from the cells were directly subjected to reducing SDS-PAGE and blotted with MAbs against phospho-EGFR (pEGFR), EGFR, and β-actin. (D) UV-PEDV incubation activates EGFR. IPEC-J2 and HEK293 cells were incubated with UV-PEDV at an MOI of 1, and the cells were then further cultured for the indicated times. The cell lysates were subjected to immunoblotting with the antibodies indicated. (E) PEDV S-Flag protein interacts with EGFR. HEK293T cells were cotransfected with PEDV S-Flag (S) and EGFR (E). At 36 h after transfection, immunoprecipitation (IP) and immunoblotting were performed as described in Materials and Methods to examine interactions between PEDV S-Flag and EGFR.
    Figure Legend Snippet: (A to C) PEDV infection induces EGFR phosphorylation. IPEC-J2, HEK293, and Vero E6 cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed with PBS, and the cells were then cultured at 37°C for different times as indicated. Mock-infected cells were used as a control. Detergent lysates collected from the cells were directly subjected to reducing SDS-PAGE and blotted with MAbs against phospho-EGFR (pEGFR), EGFR, and β-actin. (D) UV-PEDV incubation activates EGFR. IPEC-J2 and HEK293 cells were incubated with UV-PEDV at an MOI of 1, and the cells were then further cultured for the indicated times. The cell lysates were subjected to immunoblotting with the antibodies indicated. (E) PEDV S-Flag protein interacts with EGFR. HEK293T cells were cotransfected with PEDV S-Flag (S) and EGFR (E). At 36 h after transfection, immunoprecipitation (IP) and immunoblotting were performed as described in Materials and Methods to examine interactions between PEDV S-Flag and EGFR.

    Techniques Used: Infection, Incubation, Cell Culture, SDS Page, Transfection, Immunoprecipitation

    PEDV infection-induced STAT3 activation negatively regulates ISGs. (A and B) Infection with PEDV leads to STAT3 phosphorylation. IPEC-J2 (A) and HEK293 (B) cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed, and the cells were further incubated for different periods, as indicated. The cell lysates were blotted with the MAbs against phospho-STAT3 (pSTAT3), STAT3, and β-actin. The mock-infected cells were used as a control. (C and D) Inhibition of STAT3 function enhances antiviral responses. IPEC-J2 (C) and HEK293 (D) cells were treated with the STAT3-specific inhibitor S3I-201 (40 μM) for 24 h. The RNA levels of ISGs, MxA , ISG15 , and IFN-β , were determined by quantitative RT-PCR. (E) Effect of STAT3 inhibitor on cell viability. IPEC-J2 and HEK293 cells were treated with S3I-201 at the indicated concentrations or with the carrier control DMSO for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. The results are representative of three independent experiments (means and SD). *, P
    Figure Legend Snippet: PEDV infection-induced STAT3 activation negatively regulates ISGs. (A and B) Infection with PEDV leads to STAT3 phosphorylation. IPEC-J2 (A) and HEK293 (B) cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed, and the cells were further incubated for different periods, as indicated. The cell lysates were blotted with the MAbs against phospho-STAT3 (pSTAT3), STAT3, and β-actin. The mock-infected cells were used as a control. (C and D) Inhibition of STAT3 function enhances antiviral responses. IPEC-J2 (C) and HEK293 (D) cells were treated with the STAT3-specific inhibitor S3I-201 (40 μM) for 24 h. The RNA levels of ISGs, MxA , ISG15 , and IFN-β , were determined by quantitative RT-PCR. (E) Effect of STAT3 inhibitor on cell viability. IPEC-J2 and HEK293 cells were treated with S3I-201 at the indicated concentrations or with the carrier control DMSO for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. The results are representative of three independent experiments (means and SD). *, P

    Techniques Used: Infection, Activation Assay, Incubation, Inhibition, Quantitative RT-PCR, CCK-8 Assay

    Knockdown of EGFR expression decreases PEDV infection. (A and C) Verification of EGFR knockdown efficiency. IPEC-J2 (A) and HEK293 (C) cells were transfected with EGFR-specific siRNA (siEGFR) or scrambled control siRNA (siCtrl) for 24 h, and the knockdown efficiency of EGFR was determined by quantitative RT-PCR and Western blotting. (B and D) Depletion of endogenous EGFR inhibited PEDV replication. After siRNA transfection for 24 h, cells were exposed to virus for 48 h (IPEC-J2) (B) or 24 h (HEK293) (D). The effect of EGFR knockdown on PEDV infection was determined by Western blotting (A and C, right), quantitative RT-PCR, and TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P
    Figure Legend Snippet: Knockdown of EGFR expression decreases PEDV infection. (A and C) Verification of EGFR knockdown efficiency. IPEC-J2 (A) and HEK293 (C) cells were transfected with EGFR-specific siRNA (siEGFR) or scrambled control siRNA (siCtrl) for 24 h, and the knockdown efficiency of EGFR was determined by quantitative RT-PCR and Western blotting. (B and D) Depletion of endogenous EGFR inhibited PEDV replication. After siRNA transfection for 24 h, cells were exposed to virus for 48 h (IPEC-J2) (B) or 24 h (HEK293) (D). The effect of EGFR knockdown on PEDV infection was determined by Western blotting (A and C, right), quantitative RT-PCR, and TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Techniques Used: Expressing, Infection, Transfection, Quantitative RT-PCR, Western Blot

    4) Product Images from "Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function"

    Article Title: Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20187599

    Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P
    Figure Legend Snippet: Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P

    Techniques Used: Migration, Staining, Wound Healing Assay, Clonogenic Assay, CCK-8 Assay

    5) Product Images from "Epstein-Barr Virus Nuclear Antigen 1 Recruits Cyclophilin A to Facilitate the Replication of Viral DNA Genome"

    Article Title: Epstein-Barr Virus Nuclear Antigen 1 Recruits Cyclophilin A to Facilitate the Replication of Viral DNA Genome

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02879

    Effect of CYPA on EBNA1 expression and loss of viral copy numbers. (A) Designations of the CYPA siRNAs. Three siRNAs for CYPA and a control siRNA were designed and used for detection of the interference efficiency. HEK293 cells were transfected with one of the siRNAs, and a WB assay was performed at 48 h post-transfection for analysis of the CYPA protein level. (B,C) Effect of siCYPA on EBNA1 expression in EBV-positive cells. Two EBV-positive cell lines [C666-1 (B) and C2089 (C) ] were used for siRNA transfection. At 48 h post-transfection, the cell lysates and RNA were subjected to the WB and RT-qPCR assays respectively, with siCYPA standarized to 1. (D,E) Effect of CYPA overexpression on EBNA1 expression at protein and mRNA levels in EBV-positive cells [C666-1 (D) and C2089 (E) ]. (F) The effect of CYPA depletion on loss of EBV copy numbers during passages. EBV-positive C2089 was used for shRNA stable transfection and selection. Cell lines stably transfected with shRNA-CYPA or shRNA-NC were established for the experiment. Cell dispersal for each passage was performed at a ratio 1:2. The copy number of the C2089-shCYPA cells was decreased compared with the C2089-shNC, with passage 10th of C2089-shCYPA set to 1. ∗ P
    Figure Legend Snippet: Effect of CYPA on EBNA1 expression and loss of viral copy numbers. (A) Designations of the CYPA siRNAs. Three siRNAs for CYPA and a control siRNA were designed and used for detection of the interference efficiency. HEK293 cells were transfected with one of the siRNAs, and a WB assay was performed at 48 h post-transfection for analysis of the CYPA protein level. (B,C) Effect of siCYPA on EBNA1 expression in EBV-positive cells. Two EBV-positive cell lines [C666-1 (B) and C2089 (C) ] were used for siRNA transfection. At 48 h post-transfection, the cell lysates and RNA were subjected to the WB and RT-qPCR assays respectively, with siCYPA standarized to 1. (D,E) Effect of CYPA overexpression on EBNA1 expression at protein and mRNA levels in EBV-positive cells [C666-1 (D) and C2089 (E) ]. (F) The effect of CYPA depletion on loss of EBV copy numbers during passages. EBV-positive C2089 was used for shRNA stable transfection and selection. Cell lines stably transfected with shRNA-CYPA or shRNA-NC were established for the experiment. Cell dispersal for each passage was performed at a ratio 1:2. The copy number of the C2089-shCYPA cells was decreased compared with the C2089-shNC, with passage 10th of C2089-shCYPA set to 1. ∗ P

    Techniques Used: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Over Expression, shRNA, Stable Transfection, Selection

    Identification of the EBNA1 domain required for binding to CYPA. (A) Diagram of the EBNA1 deletion mutants. The start and end amino acid residues for each fragment are indicated according to a previous report ( Young and Murray, 2003 ). (B) Validation of the interaction between each mutant EBNA1 and CYPA by the co-IP assay. PCAGGS-Myc-EBNA1, pCAGGS-Myc-EBNA1 mutants, and pCAGGS-Flag-CYPA were transfected into HEK293 cells. Myc-EBNA1 was immune-precipitated with anti-FLAG antibody. Flag-CYPA and Myc-EBNA1 were detected by WB.
    Figure Legend Snippet: Identification of the EBNA1 domain required for binding to CYPA. (A) Diagram of the EBNA1 deletion mutants. The start and end amino acid residues for each fragment are indicated according to a previous report ( Young and Murray, 2003 ). (B) Validation of the interaction between each mutant EBNA1 and CYPA by the co-IP assay. PCAGGS-Myc-EBNA1, pCAGGS-Myc-EBNA1 mutants, and pCAGGS-Flag-CYPA were transfected into HEK293 cells. Myc-EBNA1 was immune-precipitated with anti-FLAG antibody. Flag-CYPA and Myc-EBNA1 were detected by WB.

    Techniques Used: Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Western Blot

    The effect of CYPA-specific inhibitor CsA on EBNA1–mediated oriP transcription and EBNA1-oriP binding. (A) The concentration determination of CsA by Cell Counting Kit-8 (CCK-8) assay. The concentration of 40 μM at 48 h was determined as the working concentration for treatment. (B) Rescue experiment of EBNA1 protein level after CsA treatment in C2089 cells. (C) The protein expression of CYPA and EBNA1 detected by WB assay at 48 h post-treatment with CsA. The mRNA expression of EBNA1 measured by RT-qPCR. EBV-positive C2089 cell lines were used for the test. (D) C2089 cells were transfected with oriP-SV40-Luc reporter plasmid and CYPA expression plasmids. Overexpressed CYPA significantly increased EBNA1- oriP-dependent luciferase activity, but had no effect on SV40 promoter dependent luciferase activity (left). EBNA1 mRNA was measured by RT-qPCR (right). (E) OriP-SV40-Luc reporter plasmid was transfected into C2089 cells, following the CsA treatment. CsA treatment greatly reduced EBNA1- oriP luciferase activity compared with untreatment. (F) CsA and elevated CYPA on EBNA1-oriP-mediated transcription activity in the luciferase reporter assay in HEK293 cells. (G) ChIP- qPCR was used to determine EBNA1-oriP binding. CsA treatment reduced EBNA1-oriP binding while elevated CYPA increased binding. CYPA and EBNA1 proteins were analyzed by WB. ∗ P
    Figure Legend Snippet: The effect of CYPA-specific inhibitor CsA on EBNA1–mediated oriP transcription and EBNA1-oriP binding. (A) The concentration determination of CsA by Cell Counting Kit-8 (CCK-8) assay. The concentration of 40 μM at 48 h was determined as the working concentration for treatment. (B) Rescue experiment of EBNA1 protein level after CsA treatment in C2089 cells. (C) The protein expression of CYPA and EBNA1 detected by WB assay at 48 h post-treatment with CsA. The mRNA expression of EBNA1 measured by RT-qPCR. EBV-positive C2089 cell lines were used for the test. (D) C2089 cells were transfected with oriP-SV40-Luc reporter plasmid and CYPA expression plasmids. Overexpressed CYPA significantly increased EBNA1- oriP-dependent luciferase activity, but had no effect on SV40 promoter dependent luciferase activity (left). EBNA1 mRNA was measured by RT-qPCR (right). (E) OriP-SV40-Luc reporter plasmid was transfected into C2089 cells, following the CsA treatment. CsA treatment greatly reduced EBNA1- oriP luciferase activity compared with untreatment. (F) CsA and elevated CYPA on EBNA1-oriP-mediated transcription activity in the luciferase reporter assay in HEK293 cells. (G) ChIP- qPCR was used to determine EBNA1-oriP binding. CsA treatment reduced EBNA1-oriP binding while elevated CYPA increased binding. CYPA and EBNA1 proteins were analyzed by WB. ∗ P

    Techniques Used: Binding Assay, Concentration Assay, Cell Counting, CCK-8 Assay, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The effect of CYPA knockdown on EBNA1–mediated oriP transcription activity. (A) The detection of CYPA expression in stably transfected cell lines with shRNA-CYPA and shRNA-NC. (B) Restoration of EBNA1 protein expression in C2089-shCYPA cells transfected with wild-type CYPA expression plasmids. CYPA and EBNA1 proteins were detected by WB. (C) Effect of CYPA knockdown on EBNA1-oriP-mediated transcription activity in the luciferase reporter assay. The EBNA1 mRNA was detected by RT-qPCR. (D) Effect of CYPA knockdown on binding of EBNA1 to oriP in the ChIP assay. CYPA was depleted from HEK293 with shRNA. Antibodies against Myc and IgG control were respectively used for the pulldown in ChIP assaysHEK293. The precipitation of oriP DNA was quantitated by RT-qPCR. CYPA and EBNA1 proteins were detected by WB. ∗ P
    Figure Legend Snippet: The effect of CYPA knockdown on EBNA1–mediated oriP transcription activity. (A) The detection of CYPA expression in stably transfected cell lines with shRNA-CYPA and shRNA-NC. (B) Restoration of EBNA1 protein expression in C2089-shCYPA cells transfected with wild-type CYPA expression plasmids. CYPA and EBNA1 proteins were detected by WB. (C) Effect of CYPA knockdown on EBNA1-oriP-mediated transcription activity in the luciferase reporter assay. The EBNA1 mRNA was detected by RT-qPCR. (D) Effect of CYPA knockdown on binding of EBNA1 to oriP in the ChIP assay. CYPA was depleted from HEK293 with shRNA. Antibodies against Myc and IgG control were respectively used for the pulldown in ChIP assaysHEK293. The precipitation of oriP DNA was quantitated by RT-qPCR. CYPA and EBNA1 proteins were detected by WB. ∗ P

    Techniques Used: Activity Assay, Expressing, Stable Transfection, Transfection, shRNA, Western Blot, Luciferase, Reporter Assay, Quantitative RT-PCR, Binding Assay, Chromatin Immunoprecipitation

    The effect of ectopic CYPA expression on USP7-EBNA1 and EBNA1-oriP binding. (A) Effect of CYPA overexpression on EBNA1-USP7 binding in HEK293-shCYPA and HEK293-shNC cells. These cells were transiently transfected with pCAGGS-Flag-CYPA alone or with pCAGGS-Myc-EBNA1, a co-IP assay was performed with the anti-Myc antibody for the pulldown. (B) The inhibition of CsA on CYPA-USP7 antagonism in the binding with EBNA1. pCAGGS-Myc-EBNA1 was transfected with pCAGGS-Flag-CYPA or alone, and treated with CsA. The anti-Myc antibody was used for the pulldown, and proteins were detected by WB. (C) Effect of CYPA overexpression and EBNA1Δ376-459 mutation on the EBNA1-oriP binding detected by ChIP assay. ∗ P
    Figure Legend Snippet: The effect of ectopic CYPA expression on USP7-EBNA1 and EBNA1-oriP binding. (A) Effect of CYPA overexpression on EBNA1-USP7 binding in HEK293-shCYPA and HEK293-shNC cells. These cells were transiently transfected with pCAGGS-Flag-CYPA alone or with pCAGGS-Myc-EBNA1, a co-IP assay was performed with the anti-Myc antibody for the pulldown. (B) The inhibition of CsA on CYPA-USP7 antagonism in the binding with EBNA1. pCAGGS-Myc-EBNA1 was transfected with pCAGGS-Flag-CYPA or alone, and treated with CsA. The anti-Myc antibody was used for the pulldown, and proteins were detected by WB. (C) Effect of CYPA overexpression and EBNA1Δ376-459 mutation on the EBNA1-oriP binding detected by ChIP assay. ∗ P

    Techniques Used: Expressing, Binding Assay, Over Expression, Transfection, Co-Immunoprecipitation Assay, Inhibition, Western Blot, Mutagenesis, Chromatin Immunoprecipitation

    Detection of CYPA-EBNA1 binding by the BiMC and co-IP assays. (A) Detection of the EBNA1-CYPA interaction by the BiMC assay. HEK293T cells were transfected with the indicated plasmids, including CYPA-NY, EBNA1-CY, EBNA1ΔNLS-CY, and CYPB-NY following by BiMC analysis, and fluorescence was observed. Transfection of a single CYPA-NY plasmid did not lead to the production of fluorescence. Scale bar, 50 μm. (B) The proteins expressed from the plasmids used in the BiMC assay were detected by WB. (C) The detection of CYPA and EBNA1 in EBV-negative and EBV-positive HEK293 cells by IF assay. Scale bar, 50 μm. (D) Endogenous CYPA interacts with EBNA1 in HEK293 cells. The plasmid pCAGGS-Myc-EBNA1 was transfected into cells. An anti-Myc antibody was used for the pull-down the CYPA, and the WB assay was carried out for detection. (E) Endogenous CYPA interacts with EBNA1 in C2089 cells. EBNA1 was immune-precipitated with anti-CYPA. EBNA1 was detected by WB (F) Exogenous CYPA interacts with EBNA1. HEK293 cells were transiently transfected with Flag-CYPA alone or with Myc-EBNA1. Flag-CYPA was immune-precipitated with anti-Myc antibody. IgG was used as a negative control for the pull-down in the co-IP assay. Flag-CYPA was detected by WB. (G) Co-IP assay for comparison of the interactions of CYPA and CYPB with EBNA1. PCAGGS-Myc-EBNA1 and pCAGGS-Flag-CYPA or pCAGGS-Flag-CYPB were transfected in HEK293 cells. Myc-EBNA1 was immune-precipitated with anti-Flag antibody.
    Figure Legend Snippet: Detection of CYPA-EBNA1 binding by the BiMC and co-IP assays. (A) Detection of the EBNA1-CYPA interaction by the BiMC assay. HEK293T cells were transfected with the indicated plasmids, including CYPA-NY, EBNA1-CY, EBNA1ΔNLS-CY, and CYPB-NY following by BiMC analysis, and fluorescence was observed. Transfection of a single CYPA-NY plasmid did not lead to the production of fluorescence. Scale bar, 50 μm. (B) The proteins expressed from the plasmids used in the BiMC assay were detected by WB. (C) The detection of CYPA and EBNA1 in EBV-negative and EBV-positive HEK293 cells by IF assay. Scale bar, 50 μm. (D) Endogenous CYPA interacts with EBNA1 in HEK293 cells. The plasmid pCAGGS-Myc-EBNA1 was transfected into cells. An anti-Myc antibody was used for the pull-down the CYPA, and the WB assay was carried out for detection. (E) Endogenous CYPA interacts with EBNA1 in C2089 cells. EBNA1 was immune-precipitated with anti-CYPA. EBNA1 was detected by WB (F) Exogenous CYPA interacts with EBNA1. HEK293 cells were transiently transfected with Flag-CYPA alone or with Myc-EBNA1. Flag-CYPA was immune-precipitated with anti-Myc antibody. IgG was used as a negative control for the pull-down in the co-IP assay. Flag-CYPA was detected by WB. (G) Co-IP assay for comparison of the interactions of CYPA and CYPB with EBNA1. PCAGGS-Myc-EBNA1 and pCAGGS-Flag-CYPA or pCAGGS-Flag-CYPB were transfected in HEK293 cells. Myc-EBNA1 was immune-precipitated with anti-Flag antibody.

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Fluorescence, Plasmid Preparation, Western Blot, Negative Control

    6) Product Images from "Silencing of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs Stimulates Hyperplastic Phenotypes through Activation of Epidermal Growth Factor Receptor and Hypoxia-Inducible Factor-2?"

    Article Title: Silencing of Reversion-Inducing Cysteine-Rich Protein with Kazal Motifs Stimulates Hyperplastic Phenotypes through Activation of Epidermal Growth Factor Receptor and Hypoxia-Inducible Factor-2?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084520

    Hypoxia and silencing of RECK increased cell cycle progression. ( A ) Western blot analysis with anti-c-Myc, cyclin D1, cyclin A, phosphorylated Rb (p-pRb), p21, p27, and p16 were performed under normoxic (N) and hypoxic (H) conditions after 24 h of exposure. ( B ) After transfecting scrambled siRNA (Scr) and two kinds of siRECK (si1 and si2), levels of cell cycle proteins in cell lysates were determined by Western blotting. ( C ) Cell cycle analysis by FACS after PI staining. The diagram shows the diploid S and sub-G1 phase HEK293 cells under hypoxic conditions up to 72 h after staining and those of normoxic controls (cont). Right cytograms show the proportion of cell populations in each cell cycle stage in 12 h normoxia (12N) and hypoxia (12H). All experiments were performed at least in duplicate.
    Figure Legend Snippet: Hypoxia and silencing of RECK increased cell cycle progression. ( A ) Western blot analysis with anti-c-Myc, cyclin D1, cyclin A, phosphorylated Rb (p-pRb), p21, p27, and p16 were performed under normoxic (N) and hypoxic (H) conditions after 24 h of exposure. ( B ) After transfecting scrambled siRNA (Scr) and two kinds of siRECK (si1 and si2), levels of cell cycle proteins in cell lysates were determined by Western blotting. ( C ) Cell cycle analysis by FACS after PI staining. The diagram shows the diploid S and sub-G1 phase HEK293 cells under hypoxic conditions up to 72 h after staining and those of normoxic controls (cont). Right cytograms show the proportion of cell populations in each cell cycle stage in 12 h normoxia (12N) and hypoxia (12H). All experiments were performed at least in duplicate.

    Techniques Used: Western Blot, Cell Cycle Assay, FACS, Staining

    Involvement of the EGFR signaling pathway in HIF-2α expression in RECK-silenced cells. ( A ) HEK293 cells were exposed hypoxia for 6, 12, and 24 h and determined time-dependent HIF-2α expression by western blot analysis (upper panel). HEK293 cells transfected with scrambled si RNA (-) or siRECK were treated with gefitinib (Ge, 1 μM, an EGFR inhibitor) or PD98059 (PD, 50 μM; an ERK MAPK inhibitor) under normoxia (N) or hypoxia (H) for 24 h, and the protein expressions of HIF-2α were determined by western blot analysis. β-actin was used for internal control. ( B ) The activations of EGFR and ERK-MAPK in scrambled (scr) or siRECK (siR) transfected cells exposed to normoxia (N) or hypoxia (H) were determined by western blotting using antibodies against total form or phosphorylated-form of EGFR or ERK. ( C ) The activations of EGFR and ERK and RECK expression in siHIF-1α (siH1) -, siHIF-2α (siH2) -, and MMP inhibitor (Mib)-treated cells under hypoxia (H) were determined by western blot analysis using antibodies against phosphorylated-EGFR and RECK antibody. Scr. Scrambled siRNA.
    Figure Legend Snippet: Involvement of the EGFR signaling pathway in HIF-2α expression in RECK-silenced cells. ( A ) HEK293 cells were exposed hypoxia for 6, 12, and 24 h and determined time-dependent HIF-2α expression by western blot analysis (upper panel). HEK293 cells transfected with scrambled si RNA (-) or siRECK were treated with gefitinib (Ge, 1 μM, an EGFR inhibitor) or PD98059 (PD, 50 μM; an ERK MAPK inhibitor) under normoxia (N) or hypoxia (H) for 24 h, and the protein expressions of HIF-2α were determined by western blot analysis. β-actin was used for internal control. ( B ) The activations of EGFR and ERK-MAPK in scrambled (scr) or siRECK (siR) transfected cells exposed to normoxia (N) or hypoxia (H) were determined by western blotting using antibodies against total form or phosphorylated-form of EGFR or ERK. ( C ) The activations of EGFR and ERK and RECK expression in siHIF-1α (siH1) -, siHIF-2α (siH2) -, and MMP inhibitor (Mib)-treated cells under hypoxia (H) were determined by western blot analysis using antibodies against phosphorylated-EGFR and RECK antibody. Scr. Scrambled siRNA.

    Techniques Used: Expressing, Western Blot, Transfection

    The involvement of HIF-2α in hypoxia-induced RECK silencing-mediated cell proliferation. ( A ) HEK293 cells were transfected with scrambled or RECK siRNA and exposed to normoxic or hypoxic conditions for 24 h. The expressions of HIF-1α HIF-2α, NFκB, and phophorylated-STAT1 were determined by western blotting. β-actin was used as an internal control. ( B ) Using the same samples as in A, the mRNA expressions of RECK and HIF-2α were determined by semi-quantitative RT-PCR. β-actin was used as an internal control. ( C ) HEK293 cells were plated onto 96-well plates and transfected with siHIF-1α and/or siHIF-2α or scrambled siRNA using HiPerFect Transfection reagent (Qiagen). After 24 h, transfected cells were incubated under normoxic or hypoxic conditions for an additional 24 h. At the indicated time points, cell proliferation assays were performed using CCK-8 solution. Data are presented as means ± SDs (n > 3). Expression of HIF-1α and HIF-2α was confirmed in each siRNA transfectant by western blot analysis (right panel). RECK, HIF-1α, HIF-2α mRNA expression pattern was confirmed by semiquantitative RT-PCR at indicated time point. β-actin was used as an internal control (lower panel). *, p
    Figure Legend Snippet: The involvement of HIF-2α in hypoxia-induced RECK silencing-mediated cell proliferation. ( A ) HEK293 cells were transfected with scrambled or RECK siRNA and exposed to normoxic or hypoxic conditions for 24 h. The expressions of HIF-1α HIF-2α, NFκB, and phophorylated-STAT1 were determined by western blotting. β-actin was used as an internal control. ( B ) Using the same samples as in A, the mRNA expressions of RECK and HIF-2α were determined by semi-quantitative RT-PCR. β-actin was used as an internal control. ( C ) HEK293 cells were plated onto 96-well plates and transfected with siHIF-1α and/or siHIF-2α or scrambled siRNA using HiPerFect Transfection reagent (Qiagen). After 24 h, transfected cells were incubated under normoxic or hypoxic conditions for an additional 24 h. At the indicated time points, cell proliferation assays were performed using CCK-8 solution. Data are presented as means ± SDs (n > 3). Expression of HIF-1α and HIF-2α was confirmed in each siRNA transfectant by western blot analysis (right panel). RECK, HIF-1α, HIF-2α mRNA expression pattern was confirmed by semiquantitative RT-PCR at indicated time point. β-actin was used as an internal control (lower panel). *, p

    Techniques Used: Transfection, Western Blot, Quantitative RT-PCR, Incubation, CCK-8 Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    RECK silencing induced by hypoxia stimulated epithelial cell proliferation. ( A ) Site and silencing efficiency of two RECK siRNAs. The upper panel shows RECK siRNA sites (red bar). The lower panel shows that transfection of RECK siRNAs (5 nM) into HEK293 cells effectively blocked RECK expression. The experiment was performed in triplicate. ( B ) Proliferation assays were performed at 24 h post-transfection (0 time) at the indicated times after seeding siRNA-transfected cells at 5x10 3 per well in 96-well plates. Fold change is the ratio of the cell population at each time point versus the start time (point 0). Data are presented as means ± SDs (n=4). *, p
    Figure Legend Snippet: RECK silencing induced by hypoxia stimulated epithelial cell proliferation. ( A ) Site and silencing efficiency of two RECK siRNAs. The upper panel shows RECK siRNA sites (red bar). The lower panel shows that transfection of RECK siRNAs (5 nM) into HEK293 cells effectively blocked RECK expression. The experiment was performed in triplicate. ( B ) Proliferation assays were performed at 24 h post-transfection (0 time) at the indicated times after seeding siRNA-transfected cells at 5x10 3 per well in 96-well plates. Fold change is the ratio of the cell population at each time point versus the start time (point 0). Data are presented as means ± SDs (n=4). *, p

    Techniques Used: Transfection, Expressing

    Silencing of RECK increased in vitro soft-agar colony formation and in vivo tumorigenesis. ( A ) HEK293 cells were transfected with si RECK and plated onto agar-coated 96-well plates 24 h after transfection. Colonies were observed one week after seeding (upper panel). Original magnification 40X. Quantitation of colony formation by HEK293 cells transfected with scrambled or RECK siRNA1 one week after seeding. Colony formation was measured using a fluorometer, as described in Materials and Methods. Data presented as means ± SDs (n=4). *, p
    Figure Legend Snippet: Silencing of RECK increased in vitro soft-agar colony formation and in vivo tumorigenesis. ( A ) HEK293 cells were transfected with si RECK and plated onto agar-coated 96-well plates 24 h after transfection. Colonies were observed one week after seeding (upper panel). Original magnification 40X. Quantitation of colony formation by HEK293 cells transfected with scrambled or RECK siRNA1 one week after seeding. Colony formation was measured using a fluorometer, as described in Materials and Methods. Data presented as means ± SDs (n=4). *, p

    Techniques Used: In Vitro, In Vivo, Transfection, Quantitation Assay

    MMP-2/-9 and MT1-MMP were also involved in hypoxia-induced epithelial cell proliferation. ( A ) Cells were transfected with scrambled siRNA or the two types of siRECKs. After 24 h, transfected cells were incubated under normoxic or hypoxic conditions for an additional 24 h. Conditioned media were collected from siRECK transfected (si1 and si2) or hypoxia-exposed HEK 293 cells, and gelatin zymography was performed. Western blotting for MT1-MMP was also performed using protein lysates obtained under each condition. ( B ) HEK293 cells were pretreated with a MMP inhibitor (5 µM) 2 h before normoxic or hypoxic incubation and cell proliferation assays were performed at the indicated times under normoxic (N) or hypoxic (H) conditions. Data are presented as means ± SDs (n=4). *, p
    Figure Legend Snippet: MMP-2/-9 and MT1-MMP were also involved in hypoxia-induced epithelial cell proliferation. ( A ) Cells were transfected with scrambled siRNA or the two types of siRECKs. After 24 h, transfected cells were incubated under normoxic or hypoxic conditions for an additional 24 h. Conditioned media were collected from siRECK transfected (si1 and si2) or hypoxia-exposed HEK 293 cells, and gelatin zymography was performed. Western blotting for MT1-MMP was also performed using protein lysates obtained under each condition. ( B ) HEK293 cells were pretreated with a MMP inhibitor (5 µM) 2 h before normoxic or hypoxic incubation and cell proliferation assays were performed at the indicated times under normoxic (N) or hypoxic (H) conditions. Data are presented as means ± SDs (n=4). *, p

    Techniques Used: Transfection, Incubation, Zymography, Western Blot

    7) Product Images from "Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function"

    Article Title: Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20187599

    Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P
    Figure Legend Snippet: Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P

    Techniques Used: Migration, Staining, Wound Healing Assay, Clonogenic Assay, CCK-8 Assay

    8) Product Images from "Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT)"

    Article Title: Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT)

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2021.103240

    Oncolysis and replication of VVs in tumor cells. (A) 4T1, CT26, MC38, and H22 cells were plated into 96-well plates and infected with VVs at the indicated MOI for 48 and 72 h. The cell viability was determined by MTT assay for 4T1, CT26, and MC38 cells and was determined by CCK8 assay for H22 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. (B) 4T1, CT26, and MC38 cells were plated into a 96-well plate and infected with VVs at the indicated MOI for 72 h, and oncolytic ability was determined by crystal violet staining. The figure represents one of the three experiments performed. ( C ) 4T1, CT26, MC38, and H22 cells were infected with VVs at MOI of 0.1, cells were harvested at designated time points and progeny viral particles were quantified by titration assays (TCID50).
    Figure Legend Snippet: Oncolysis and replication of VVs in tumor cells. (A) 4T1, CT26, MC38, and H22 cells were plated into 96-well plates and infected with VVs at the indicated MOI for 48 and 72 h. The cell viability was determined by MTT assay for 4T1, CT26, and MC38 cells and was determined by CCK8 assay for H22 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. (B) 4T1, CT26, and MC38 cells were plated into a 96-well plate and infected with VVs at the indicated MOI for 72 h, and oncolytic ability was determined by crystal violet staining. The figure represents one of the three experiments performed. ( C ) 4T1, CT26, MC38, and H22 cells were infected with VVs at MOI of 0.1, cells were harvested at designated time points and progeny viral particles were quantified by titration assays (TCID50).

    Techniques Used: Infection, MTT Assay, CCK-8 Assay, Standard Deviation, Staining, Titration

    Expression of PVR/CD155 and PD-L1 on tumor cells. Flow cytometry histogram representative of the expression of PVR/CD155 (A) and PD-L1 (B) on 4T1, CT26, MC38 and H22 cells. To analyze PVR/CD155, PE-conjugated rat-anti-mouse IgG2a was used as an isotype control. To analyze PD-L1, PE-conjugated rat-anti-mouse IgG2b was used as an isotype control. Data are presented as the mean fluorescence intensity (MFI) ± SD of three independent assay. ** P
    Figure Legend Snippet: Expression of PVR/CD155 and PD-L1 on tumor cells. Flow cytometry histogram representative of the expression of PVR/CD155 (A) and PD-L1 (B) on 4T1, CT26, MC38 and H22 cells. To analyze PVR/CD155, PE-conjugated rat-anti-mouse IgG2a was used as an isotype control. To analyze PD-L1, PE-conjugated rat-anti-mouse IgG2b was used as an isotype control. Data are presented as the mean fluorescence intensity (MFI) ± SD of three independent assay. ** P

    Techniques Used: Expressing, Flow Cytometry, Fluorescence

    Anti-tumor activity of the recombinant VV on colon cancer models. (A) Treatment scheme of MC38 tumor model. The subcutaneous tumor model was established by inoculation of 2 × 10 6 MC38 cells on the right flank of C57BL/6 mice. Mice were administered intratumorally at the indicated day for 3 times with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (C) Kaplan-Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (E) Treatment scheme of CT26 tumor model. The subcutaneous tumor model was established by inoculation of 5 × 10 5 CT26 cells on the right flank of BALB/c mice. Mice treatment were similar to ( A ). (F) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (I, J) Immunohistochemistry (IHC) detection of the infiltration of CD8 + T cells (I) and microvascular density (MVD) (J) of the tumor in the CT26 subcutaneous tumor model. The tumor model was established as previously described. When the tumor reached approximately 50 mm 3 , mice were treated i.t. with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. Seven days after VV injection, tumors were collected from mice, and CD8 and CD31 expression were detected by IHC. (K, L) The α-TIGIT levels in tumor and blood. Samples of the tumor lysates and sera were prepared as previously described in materials and methods and a luciferase assay was used to detect the levels of secreted α-TIGIT. (M) Viral titers in tumor and blood. Samples were collected similar to (K, L) , and viral titers were quantified by a TCID50 method. Statistical differences in tumor volume, body weight, CD8 + T cells, MVD, luciferase activity, and viral titer among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; * P
    Figure Legend Snippet: Anti-tumor activity of the recombinant VV on colon cancer models. (A) Treatment scheme of MC38 tumor model. The subcutaneous tumor model was established by inoculation of 2 × 10 6 MC38 cells on the right flank of C57BL/6 mice. Mice were administered intratumorally at the indicated day for 3 times with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (C) Kaplan-Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (E) Treatment scheme of CT26 tumor model. The subcutaneous tumor model was established by inoculation of 5 × 10 5 CT26 cells on the right flank of BALB/c mice. Mice treatment were similar to ( A ). (F) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (I, J) Immunohistochemistry (IHC) detection of the infiltration of CD8 + T cells (I) and microvascular density (MVD) (J) of the tumor in the CT26 subcutaneous tumor model. The tumor model was established as previously described. When the tumor reached approximately 50 mm 3 , mice were treated i.t. with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. Seven days after VV injection, tumors were collected from mice, and CD8 and CD31 expression were detected by IHC. (K, L) The α-TIGIT levels in tumor and blood. Samples of the tumor lysates and sera were prepared as previously described in materials and methods and a luciferase assay was used to detect the levels of secreted α-TIGIT. (M) Viral titers in tumor and blood. Samples were collected similar to (K, L) , and viral titers were quantified by a TCID50 method. Statistical differences in tumor volume, body weight, CD8 + T cells, MVD, luciferase activity, and viral titer among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; * P

    Techniques Used: Activity Assay, Recombinant, Mouse Assay, Immunohistochemistry, Injection, Expressing, Luciferase

    9) Product Images from "Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway"

    Article Title: Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway

    Journal: Journal of Cancer

    doi: 10.7150/jca.13289

    Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 10 5 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P
    Figure Legend Snippet: Knockdown of hERG1 reduces proliferation of osteosarcoma cells. (A) Efficiency of knockdown by hERG1-siRNA was measured by Western blot. (B-E) Proliferation of MG-63 cells transfected with hERG1-siRNA (30 nM) (B and C), or treated with hERG1 inhibitor E-4031 (D) or activator PD 118057 (E) was measured using CCK-8 or colony formation assay (n = 6). (F) Protein expression of hERG1 in HEK293-wt and HEK293-hERG1 cells was detected by Western blot. (G) 1 × 10 5 HEK293-wt and HEK293-hERG1 cells were cultured for 48 h and the CCK-8 assay was performed. (H) The effects of E-4031 on the proliferation of HEK293-wt and HEK293-hERG1 cells were determined by CCK-8 assay. * P

    Techniques Used: Western Blot, Transfection, CCK-8 Assay, Colony Assay, Expressing, Cell Culture

    Highly expression of hERG1 in osteosarcoma cells and tissues. (A) Semi-quantitative RT-PCR was performed to measure hERG1 mRNA levels in SH-SY5Y, MG-63 and HEK293 cells. β-actin was used as the internal standard. (B) Protein expression of hERG1 was measured in SH-SY5Y, MG-63, and HEK293 cells by Western blot. GAPDH was used as the internal standard. (C) Immunohistochemistry staining for hERG1 in MG-63 cells. Images were captured using an Olympus light microscope equipped with a CCD color camera. (D) Immunohistochemistry of hERG1 in human brain (as positive control), osteosarcoma and osteosfibrous dysplasia samples. *** P
    Figure Legend Snippet: Highly expression of hERG1 in osteosarcoma cells and tissues. (A) Semi-quantitative RT-PCR was performed to measure hERG1 mRNA levels in SH-SY5Y, MG-63 and HEK293 cells. β-actin was used as the internal standard. (B) Protein expression of hERG1 was measured in SH-SY5Y, MG-63, and HEK293 cells by Western blot. GAPDH was used as the internal standard. (C) Immunohistochemistry staining for hERG1 in MG-63 cells. Images were captured using an Olympus light microscope equipped with a CCD color camera. (D) Immunohistochemistry of hERG1 in human brain (as positive control), osteosarcoma and osteosfibrous dysplasia samples. *** P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Staining, Light Microscopy, Positive Control

    10) Product Images from "Translation of the FMR1 mRNA is not influenced by AGG interruptions"

    Article Title: Translation of the FMR1 mRNA is not influenced by AGG interruptions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp713

    HEK-293 cell transfections of pRL-CMV and either pCMV- FMR1 (0AGG)-FL, pCMV- FMR1 (1AGG)-FL or pCMV- FMR1 (2AGG)-FL. ( A ) Normalized FL protein values. Error bars are standard deviations of six replicate experiments. ( B ) Normalized FL mRNA values. Error bars are standard deviations of three replicate experiments. ( C ) Normalized FL protein:mRNA ratios. Error bars are standard deviations of three replicate experiments.
    Figure Legend Snippet: HEK-293 cell transfections of pRL-CMV and either pCMV- FMR1 (0AGG)-FL, pCMV- FMR1 (1AGG)-FL or pCMV- FMR1 (2AGG)-FL. ( A ) Normalized FL protein values. Error bars are standard deviations of six replicate experiments. ( B ) Normalized FL mRNA values. Error bars are standard deviations of three replicate experiments. ( C ) Normalized FL protein:mRNA ratios. Error bars are standard deviations of three replicate experiments.

    Techniques Used: Transfection

    11) Product Images from "Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity"

    Article Title: Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

    Journal: Nature chemical biology

    doi: 10.1038/s41589-021-00878-4

    Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.
    Figure Legend Snippet: Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.

    Techniques Used: Mass Spectrometry, CRISPR, Serial Dilution, Concentration Assay, Expressing, Two Tailed Test

    SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL. a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC 50 values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC 50 values for each compound are shown for both permeabilized and live ce lls. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.
    Figure Legend Snippet: SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL. a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC 50 values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC 50 values for each compound are shown for both permeabilized and live ce lls. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.

    Techniques Used: Size-exclusion Chromatography, Incubation, Mutagenesis, Biosensor Assay, Transfection, Serial Dilution, Concentration Assay, Binding Assay, Titration, Expressing, Variant Assay

    Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader. a,b) Inspection of ternary complex crystal structures of VHL:MZ1:BRD4 BD2 (a, PDB:5T35) and BRD4 BD2 :MT1:BRD4 BD2 (b, PDB 5JWM) guided the identification of solvent-exposed region for chemical branching of linkers in trivalent PROTAC design. Chemical structures of parent bivalent molecules MZ1 and MT1 are shown. c) Chemical structures of designed trivalent PROTACs SIM1-6 based on VHL and CRBN E3 ligase ligands. d) Immunoblot analysis of BRD2, BRD3, BRD4 after treatment of HEK293 cells with 1μM PROTACs or DMSO for 4h, performed as n=1. Full blots are supplied as Source Data Fig. 1 . e) Protein levels of BRD2, BRD3, BRD4 in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 for 4h. Quantification of BET protein levels was done relative to DMSO control and shown plots used to measure the tabulated DC 50 values. Corresponding blots are in Extended Data Fig. 1c , and full blots are supplied as Source Data Fig. 1 . f) Cell viability of MV4;11 AML cell line following treatment with PROTACs or DMSO for 48h in three replicates for each concentration point. g) Chemical structures of SIM1 and its designed negative controls, ( R,S )-SIM1 and cis-SIM1. Reversed stereocenters are indicated by asterisks. h) Immunoblot of degradation of BET proteins in HEK293 cells after treatment with indicated compounds at 1μM or DMSO for 4h. Full blots are supplied as Source Data Fig. 1 . i) CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells were treated with 100nM of DMSO, MZ1, ( R,S )-SIM1, and both 10nM and 100nM of SIM1 in replicate plates for washout experiments. Media containing the 10nM and/or 100nM compounds was removed at 3.5h, indicated on the graphs, and replaced with media lacking compounds for the remainder of the experiment. Luminescence (RLU) was continuously monitored over a 50h time period and is plotted normalized to the DMSO control as Fractional RLU.
    Figure Legend Snippet: Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader. a,b) Inspection of ternary complex crystal structures of VHL:MZ1:BRD4 BD2 (a, PDB:5T35) and BRD4 BD2 :MT1:BRD4 BD2 (b, PDB 5JWM) guided the identification of solvent-exposed region for chemical branching of linkers in trivalent PROTAC design. Chemical structures of parent bivalent molecules MZ1 and MT1 are shown. c) Chemical structures of designed trivalent PROTACs SIM1-6 based on VHL and CRBN E3 ligase ligands. d) Immunoblot analysis of BRD2, BRD3, BRD4 after treatment of HEK293 cells with 1μM PROTACs or DMSO for 4h, performed as n=1. Full blots are supplied as Source Data Fig. 1 . e) Protein levels of BRD2, BRD3, BRD4 in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 for 4h. Quantification of BET protein levels was done relative to DMSO control and shown plots used to measure the tabulated DC 50 values. Corresponding blots are in Extended Data Fig. 1c , and full blots are supplied as Source Data Fig. 1 . f) Cell viability of MV4;11 AML cell line following treatment with PROTACs or DMSO for 48h in three replicates for each concentration point. g) Chemical structures of SIM1 and its designed negative controls, ( R,S )-SIM1 and cis-SIM1. Reversed stereocenters are indicated by asterisks. h) Immunoblot of degradation of BET proteins in HEK293 cells after treatment with indicated compounds at 1μM or DMSO for 4h. Full blots are supplied as Source Data Fig. 1 . i) CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells were treated with 100nM of DMSO, MZ1, ( R,S )-SIM1, and both 10nM and 100nM of SIM1 in replicate plates for washout experiments. Media containing the 10nM and/or 100nM compounds was removed at 3.5h, indicated on the graphs, and replaced with media lacking compounds for the remainder of the experiment. Luminescence (RLU) was continuously monitored over a 50h time period and is plotted normalized to the DMSO control as Fractional RLU.

    Techniques Used: Concentration Assay, CRISPR

    12) Product Images from "Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT)"

    Article Title: Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT)

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2021.103240

    Oncolysis and replication of VVs in tumor cells. (A) 4T1, CT26, MC38, and H22 cells were plated into 96-well plates and infected with VVs at the indicated MOI for 48 and 72 h. The cell viability was determined by MTT assay for 4T1, CT26, and MC38 cells and was determined by CCK8 assay for H22 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. (B) 4T1, CT26, and MC38 cells were plated into a 96-well plate and infected with VVs at the indicated MOI for 72 h, and oncolytic ability was determined by crystal violet staining. The figure represents one of the three experiments performed. ( C ) 4T1, CT26, MC38, and H22 cells were infected with VVs at MOI of 0.1, cells were harvested at designated time points and progeny viral particles were quantified by titration assays (TCID50).
    Figure Legend Snippet: Oncolysis and replication of VVs in tumor cells. (A) 4T1, CT26, MC38, and H22 cells were plated into 96-well plates and infected with VVs at the indicated MOI for 48 and 72 h. The cell viability was determined by MTT assay for 4T1, CT26, and MC38 cells and was determined by CCK8 assay for H22 cells. Data represent the mean ± standard deviation (SD) of three independent experiments. (B) 4T1, CT26, and MC38 cells were plated into a 96-well plate and infected with VVs at the indicated MOI for 72 h, and oncolytic ability was determined by crystal violet staining. The figure represents one of the three experiments performed. ( C ) 4T1, CT26, MC38, and H22 cells were infected with VVs at MOI of 0.1, cells were harvested at designated time points and progeny viral particles were quantified by titration assays (TCID50).

    Techniques Used: Infection, MTT Assay, CCK-8 Assay, Standard Deviation, Staining, Titration

    Expression of PVR/CD155 and PD-L1 on tumor cells. Flow cytometry histogram representative of the expression of PVR/CD155 (A) and PD-L1 (B) on 4T1, CT26, MC38 and H22 cells. To analyze PVR/CD155, PE-conjugated rat-anti-mouse IgG2a was used as an isotype control. To analyze PD-L1, PE-conjugated rat-anti-mouse IgG2b was used as an isotype control. Data are presented as the mean fluorescence intensity (MFI) ± SD of three independent assay. ** P
    Figure Legend Snippet: Expression of PVR/CD155 and PD-L1 on tumor cells. Flow cytometry histogram representative of the expression of PVR/CD155 (A) and PD-L1 (B) on 4T1, CT26, MC38 and H22 cells. To analyze PVR/CD155, PE-conjugated rat-anti-mouse IgG2a was used as an isotype control. To analyze PD-L1, PE-conjugated rat-anti-mouse IgG2b was used as an isotype control. Data are presented as the mean fluorescence intensity (MFI) ± SD of three independent assay. ** P

    Techniques Used: Expressing, Flow Cytometry, Fluorescence

    Anti-tumor activity of the recombinant VV on colon cancer models. (A) Treatment scheme of MC38 tumor model. The subcutaneous tumor model was established by inoculation of 2 × 10 6 MC38 cells on the right flank of C57BL/6 mice. Mice were administered intratumorally at the indicated day for 3 times with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (C) Kaplan-Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (E) Treatment scheme of CT26 tumor model. The subcutaneous tumor model was established by inoculation of 5 × 10 5 CT26 cells on the right flank of BALB/c mice. Mice treatment were similar to ( A ). (F) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (I, J) Immunohistochemistry (IHC) detection of the infiltration of CD8 + T cells (I) and microvascular density (MVD) (J) of the tumor in the CT26 subcutaneous tumor model. The tumor model was established as previously described. When the tumor reached approximately 50 mm 3 , mice were treated i.t. with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. Seven days after VV injection, tumors were collected from mice, and CD8 and CD31 expression were detected by IHC. (K, L) The α-TIGIT levels in tumor and blood. Samples of the tumor lysates and sera were prepared as previously described in materials and methods and a luciferase assay was used to detect the levels of secreted α-TIGIT. (M) Viral titers in tumor and blood. Samples were collected similar to (K, L) , and viral titers were quantified by a TCID50 method. Statistical differences in tumor volume, body weight, CD8 + T cells, MVD, luciferase activity, and viral titer among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; * P
    Figure Legend Snippet: Anti-tumor activity of the recombinant VV on colon cancer models. (A) Treatment scheme of MC38 tumor model. The subcutaneous tumor model was established by inoculation of 2 × 10 6 MC38 cells on the right flank of C57BL/6 mice. Mice were administered intratumorally at the indicated day for 3 times with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. (B) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (C) Kaplan-Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (D) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (E) Treatment scheme of CT26 tumor model. The subcutaneous tumor model was established by inoculation of 5 × 10 5 CT26 cells on the right flank of BALB/c mice. Mice treatment were similar to ( A ). (F) Tumor volumes were determined every two days. Data are presented as the mean ± SD of 8 mice for all groups. (G) Kaplan–Meier survival curves of tumor-bearing mice treated with PBS, VV-Control, and VV-α-TIGIT. (H) The body weight of the mice. Data are presented as the mean ± SD of 8 mice for all groups. (I, J) Immunohistochemistry (IHC) detection of the infiltration of CD8 + T cells (I) and microvascular density (MVD) (J) of the tumor in the CT26 subcutaneous tumor model. The tumor model was established as previously described. When the tumor reached approximately 50 mm 3 , mice were treated i.t. with PBS, 1 × 10 7 PFU of VV-Control or VV-α-TIGIT. Seven days after VV injection, tumors were collected from mice, and CD8 and CD31 expression were detected by IHC. (K, L) The α-TIGIT levels in tumor and blood. Samples of the tumor lysates and sera were prepared as previously described in materials and methods and a luciferase assay was used to detect the levels of secreted α-TIGIT. (M) Viral titers in tumor and blood. Samples were collected similar to (K, L) , and viral titers were quantified by a TCID50 method. Statistical differences in tumor volume, body weight, CD8 + T cells, MVD, luciferase activity, and viral titer among the groups were evaluated using ANOVA. Statistical differences in survival among the groups were evaluated using the Log-Rank test. ns, no significant differences; * P

    Techniques Used: Activity Assay, Recombinant, Mouse Assay, Immunohistochemistry, Injection, Expressing, Luciferase

    13) Product Images from "Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function"

    Article Title: Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function

    Journal: Brazilian Journal of Medical and Biological Research

    doi: 10.1590/1414-431X20187599

    Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P
    Figure Legend Snippet: Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P

    Techniques Used: Migration, Staining, Wound Healing Assay, Clonogenic Assay, CCK-8 Assay

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    Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 <t>HEK293</t> cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.
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    Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.

    Journal: Nature chemical biology

    Article Title: Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

    doi: 10.1038/s41589-021-00878-4

    Figure Lengend Snippet: Quantitative degradation, ubiquitination and mass spectrometry analyses reveals SIM1 has preference for BRD2. a) Quantitative live-cell degradation kinetics of CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells following treatment with DMSO and a 3-fold serial dilution of SIM1 over concentration range of 10pM-10nM (left) or 40nM-30μM (right). Luminescence (RLU) was continuously monitored over a 22h time period and is plotted normalized to the DMSO control as Fractional RLU. Data are presented as mean values with error bars representing the SD of technical quadruplicates. b) Plots of degradation rate and % Degradation or degradation maximum (Dmax) versus concentration of SIM1 from BRD2, BRD3, and BRD4 kinetic profiles shown in (a, left) and resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. Plots of initial degradation rate versus concentration of SIM1 (40nM-30μM) for BRD2, BRD3, and BRD4 are in Extended Data Fig. 2a . c) Comparison plots of BRD2 degradation rate and degradation maximum (Dmax) versus concentration calculated from kinetic graphs of SIM1 ( Fig. 2a , left) ARV-771, ( Extended Data Fig. 2b ), and the previously determined MZ1 23 . Resulting degradation rate plateau, λmax, and Dmax 50 values from each graph are shown below. d) NanoBRET live cell ubiquitination kinetics of HiBiT-BET HEK293 cells expressing LgBiT and HaloTag-Ubiquitin following 10nM SIM1 treatment (BRD2,3,4, left) or 100nM SIM1 or MZ1 (BRD2, right). Kinetic ubiquitination profiles for 100nM SIM1 and MZ1 treatment of BRD3 and 4 are shown in Extended Data Fig. 2d . Values are expressed as fold increase over DMSO control, and error bars reflect a mean ± s.d. from quadruplicates. e) Effects of SIM1 (blue) and cis-SIM1 (red) on the proteome of MV4;11 cells treated with compound at 10nM for 4 h. Data plotted log2 of the normalized fold change in abundance against –log10 of the P value per protein from three independent experiments. All t -tests performed were two-tailed assuming equal variances. Quantification of representative proteins can be found in Extended Data Fig. 2e . Further details are in the associated Supplementary Data Set 1.

    Article Snippet: For full transient NanoBRET experiments with NanoLuc-BRD4 WT, N433F, or N140F mutants, HEK293 cells (8 ×105 ) were co-transfected with 0.02μg NanoLuc-BRD4 and 2μg of HaloTag-VHL vectors.

    Techniques: Mass Spectrometry, CRISPR, Serial Dilution, Concentration Assay, Expressing, Two Tailed Test

    SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL. a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC 50 values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC 50 values for each compound are shown for both permeabilized and live ce lls. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.

    Journal: Nature chemical biology

    Article Title: Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

    doi: 10.1038/s41589-021-00878-4

    Figure Lengend Snippet: SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL. a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC 50 values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC 50 values for each compound are shown for both permeabilized and live ce lls. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.

    Article Snippet: For full transient NanoBRET experiments with NanoLuc-BRD4 WT, N433F, or N140F mutants, HEK293 cells (8 ×105 ) were co-transfected with 0.02μg NanoLuc-BRD4 and 2μg of HaloTag-VHL vectors.

    Techniques: Size-exclusion Chromatography, Incubation, Mutagenesis, Biosensor Assay, Transfection, Serial Dilution, Concentration Assay, Binding Assay, Titration, Expressing, Variant Assay

    Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader. a,b) Inspection of ternary complex crystal structures of VHL:MZ1:BRD4 BD2 (a, PDB:5T35) and BRD4 BD2 :MT1:BRD4 BD2 (b, PDB 5JWM) guided the identification of solvent-exposed region for chemical branching of linkers in trivalent PROTAC design. Chemical structures of parent bivalent molecules MZ1 and MT1 are shown. c) Chemical structures of designed trivalent PROTACs SIM1-6 based on VHL and CRBN E3 ligase ligands. d) Immunoblot analysis of BRD2, BRD3, BRD4 after treatment of HEK293 cells with 1μM PROTACs or DMSO for 4h, performed as n=1. Full blots are supplied as Source Data Fig. 1 . e) Protein levels of BRD2, BRD3, BRD4 in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 for 4h. Quantification of BET protein levels was done relative to DMSO control and shown plots used to measure the tabulated DC 50 values. Corresponding blots are in Extended Data Fig. 1c , and full blots are supplied as Source Data Fig. 1 . f) Cell viability of MV4;11 AML cell line following treatment with PROTACs or DMSO for 48h in three replicates for each concentration point. g) Chemical structures of SIM1 and its designed negative controls, ( R,S )-SIM1 and cis-SIM1. Reversed stereocenters are indicated by asterisks. h) Immunoblot of degradation of BET proteins in HEK293 cells after treatment with indicated compounds at 1μM or DMSO for 4h. Full blots are supplied as Source Data Fig. 1 . i) CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells were treated with 100nM of DMSO, MZ1, ( R,S )-SIM1, and both 10nM and 100nM of SIM1 in replicate plates for washout experiments. Media containing the 10nM and/or 100nM compounds was removed at 3.5h, indicated on the graphs, and replaced with media lacking compounds for the remainder of the experiment. Luminescence (RLU) was continuously monitored over a 50h time period and is plotted normalized to the DMSO control as Fractional RLU.

    Journal: Nature chemical biology

    Article Title: Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

    doi: 10.1038/s41589-021-00878-4

    Figure Lengend Snippet: Structure-inspired design of trivalent PROTACs identifies VHL-based SIM1 as the most potent BET degrader. a,b) Inspection of ternary complex crystal structures of VHL:MZ1:BRD4 BD2 (a, PDB:5T35) and BRD4 BD2 :MT1:BRD4 BD2 (b, PDB 5JWM) guided the identification of solvent-exposed region for chemical branching of linkers in trivalent PROTAC design. Chemical structures of parent bivalent molecules MZ1 and MT1 are shown. c) Chemical structures of designed trivalent PROTACs SIM1-6 based on VHL and CRBN E3 ligase ligands. d) Immunoblot analysis of BRD2, BRD3, BRD4 after treatment of HEK293 cells with 1μM PROTACs or DMSO for 4h, performed as n=1. Full blots are supplied as Source Data Fig. 1 . e) Protein levels of BRD2, BRD3, BRD4 in HEK293 cells treated with serially diluted PROTACs SIM1-SIM3 for 4h. Quantification of BET protein levels was done relative to DMSO control and shown plots used to measure the tabulated DC 50 values. Corresponding blots are in Extended Data Fig. 1c , and full blots are supplied as Source Data Fig. 1 . f) Cell viability of MV4;11 AML cell line following treatment with PROTACs or DMSO for 48h in three replicates for each concentration point. g) Chemical structures of SIM1 and its designed negative controls, ( R,S )-SIM1 and cis-SIM1. Reversed stereocenters are indicated by asterisks. h) Immunoblot of degradation of BET proteins in HEK293 cells after treatment with indicated compounds at 1μM or DMSO for 4h. Full blots are supplied as Source Data Fig. 1 . i) CRISPR HiBiT-BRD2, BRD3, and BRD4 HEK293 cells were treated with 100nM of DMSO, MZ1, ( R,S )-SIM1, and both 10nM and 100nM of SIM1 in replicate plates for washout experiments. Media containing the 10nM and/or 100nM compounds was removed at 3.5h, indicated on the graphs, and replaced with media lacking compounds for the remainder of the experiment. Luminescence (RLU) was continuously monitored over a 50h time period and is plotted normalized to the DMSO control as Fractional RLU.

    Article Snippet: For full transient NanoBRET experiments with NanoLuc-BRD4 WT, N433F, or N140F mutants, HEK293 cells (8 ×105 ) were co-transfected with 0.02μg NanoLuc-BRD4 and 2μg of HaloTag-VHL vectors.

    Techniques: Concentration Assay, CRISPR

    Effect of oncogenic EML4-ALK tyrosine kinase on the biological behaviors of lung cancer cells. a EML4-ALK levels in transfected H2228 and HEK293 cells. b Increased apoptosis in H2228 cells following EML4-ALK knockdown measured by flow cytometry. c Cell viability, measured by the CCK8 assay, was increased by EML4-ALK expression in HEK293 cells. d – e Viability and proliferation of H2228 cells decreased after knockdown of EML4-ALK or STAT6. f H2228 cell viability following treatment with ALK or JAK-STAT pathway inhibitors as measured by the CCK-8 assay. * P

    Journal: BMC Pulmonary Medicine

    Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

    doi: 10.1186/s12890-021-01553-z

    Figure Lengend Snippet: Effect of oncogenic EML4-ALK tyrosine kinase on the biological behaviors of lung cancer cells. a EML4-ALK levels in transfected H2228 and HEK293 cells. b Increased apoptosis in H2228 cells following EML4-ALK knockdown measured by flow cytometry. c Cell viability, measured by the CCK8 assay, was increased by EML4-ALK expression in HEK293 cells. d – e Viability and proliferation of H2228 cells decreased after knockdown of EML4-ALK or STAT6. f H2228 cell viability following treatment with ALK or JAK-STAT pathway inhibitors as measured by the CCK-8 assay. * P

    Article Snippet: Cell culture, cytokines, and inhibitors Human embryo kidney (HEK293) cells do not express STAT6 and the human lung adenocarcinoma cell line H2228, expressing STAT6 and harboring the EML4-ALK variant 3 fusion gene, were obtained from the American Type Culture Collection.

    Techniques: Transfection, Flow Cytometry, CCK-8 Assay, Expressing

    Microarray analysis of H2228 cells without or with EML4-ALK knockdown and of HEK293 cells expressing or not the EML4-ALK variant 3. a The heat map shows the regulation of a number of JAK2-STAT pathway genes. b Expression of JAK2-STAT pathway-related genes measured by real-time PCR

    Journal: BMC Pulmonary Medicine

    Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

    doi: 10.1186/s12890-021-01553-z

    Figure Lengend Snippet: Microarray analysis of H2228 cells without or with EML4-ALK knockdown and of HEK293 cells expressing or not the EML4-ALK variant 3. a The heat map shows the regulation of a number of JAK2-STAT pathway genes. b Expression of JAK2-STAT pathway-related genes measured by real-time PCR

    Article Snippet: Cell culture, cytokines, and inhibitors Human embryo kidney (HEK293) cells do not express STAT6 and the human lung adenocarcinoma cell line H2228, expressing STAT6 and harboring the EML4-ALK variant 3 fusion gene, were obtained from the American Type Culture Collection.

    Techniques: Microarray, Expressing, Variant Assay, Real-time Polymerase Chain Reaction

    Interactions between components of the JAK2-STAT pathway and EML4-ALK. a Active STAT6 and JAK2 colocalized with ALK in EML4-ALK-transfected HEK293 cells and in H2228 cells. b : ALK and phosphorylated STAT6 protein were co-immunoprecipitated with JAK2 in EML4-ALK-transfected HEK293 cells

    Journal: BMC Pulmonary Medicine

    Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

    doi: 10.1186/s12890-021-01553-z

    Figure Lengend Snippet: Interactions between components of the JAK2-STAT pathway and EML4-ALK. a Active STAT6 and JAK2 colocalized with ALK in EML4-ALK-transfected HEK293 cells and in H2228 cells. b : ALK and phosphorylated STAT6 protein were co-immunoprecipitated with JAK2 in EML4-ALK-transfected HEK293 cells

    Article Snippet: Cell culture, cytokines, and inhibitors Human embryo kidney (HEK293) cells do not express STAT6 and the human lung adenocarcinoma cell line H2228, expressing STAT6 and harboring the EML4-ALK variant 3 fusion gene, were obtained from the American Type Culture Collection.

    Techniques: Transfection, Immunoprecipitation

    EML4-ALK transfection activated JAK2, STAT1, STAT3, STAT5, and STAT6 in HEK293 and NIH3T3 cells

    Journal: BMC Pulmonary Medicine

    Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

    doi: 10.1186/s12890-021-01553-z

    Figure Lengend Snippet: EML4-ALK transfection activated JAK2, STAT1, STAT3, STAT5, and STAT6 in HEK293 and NIH3T3 cells

    Article Snippet: Cell culture, cytokines, and inhibitors Human embryo kidney (HEK293) cells do not express STAT6 and the human lung adenocarcinoma cell line H2228, expressing STAT6 and harboring the EML4-ALK variant 3 fusion gene, were obtained from the American Type Culture Collection.

    Techniques: Transfection

    Overexpression of EGFR facilitates PEDV infection. (A and C) IPEC-J2 (A) and HEK293 (C) cells were transfected with pAAV/EGFR and pAAV vector control (con) for 24 h. The cells were then infected with PEDV for 48 h or 24 h. Detergent lysates were subjected to immunoblotting with the indicated antibodies. (B and D) EGFR overexpression enhances the levels of PEDV RNA. Total RNA was extracted from IPEC-J2 cells at 48 h postinfection (hpi) (B) or from HEK293 cells at 24 hpi (D) and analyzed by quantitative RT-PCR. (E and F) EGFR overexpression promotes PEDV titers. Virus samples were collected from IPEC-J2 cells at 48 hpi (E) or from HEK293 cells at 24 hpi (F) and measured by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: Overexpression of EGFR facilitates PEDV infection. (A and C) IPEC-J2 (A) and HEK293 (C) cells were transfected with pAAV/EGFR and pAAV vector control (con) for 24 h. The cells were then infected with PEDV for 48 h or 24 h. Detergent lysates were subjected to immunoblotting with the indicated antibodies. (B and D) EGFR overexpression enhances the levels of PEDV RNA. Total RNA was extracted from IPEC-J2 cells at 48 h postinfection (hpi) (B) or from HEK293 cells at 24 hpi (D) and analyzed by quantitative RT-PCR. (E and F) EGFR overexpression promotes PEDV titers. Virus samples were collected from IPEC-J2 cells at 48 hpi (E) or from HEK293 cells at 24 hpi (F) and measured by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Over Expression, Infection, Transfection, Plasmid Preparation, Quantitative RT-PCR

    STAT3 is involved in PEDV infection. (A and B) Reduction of virus loads by S3I-201 is concentration dependent. IPEC-J2 (A) and HEK293 (B) cells were treated with different concentrations of the STAT3 inhibitor S3I-201 for 24 h, followed by infection with PEDV. At 48 hpi (IPEC-J2) or 24 hpi (HEK293), virus infection was determined by Western blotting with antibodies to PEDV N protein and β-actin (loading control). Densitometric data for the PEDV N protein/actin ratio from three independent experiments are expressed as means and SD. Virus RNA levels were also assessed by quantitative RT-PCR. (C and D) Knockdown of STAT3 expression reduced PEDV infection. Cells were transfected with STAT3-specific siRNA for 24 h and then infected with PEDV for 48 h or 24 h. STAT3 knockdown efficiency was verified by quantitative RT-PCR and Western blotting. The effect of STAT3 knockdown on virus loads was determined by Western blotting and quantitative RT-PCR. (E and F) Overexpression of STAT3 increased PEDV infection. IPEC-J2 (E) and HEK293 (F) cells were transfected with pAAV/STAT3 or vector control. Twenty-four hours later, the cells were incubated with PEDV for an additional 48 h or 24 h. The levels of STAT3 protein and PEDV N protein were analyzed by Western blotting. Total RNA was also extracted from the cells, and the viral RNA levels were then assessed by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: STAT3 is involved in PEDV infection. (A and B) Reduction of virus loads by S3I-201 is concentration dependent. IPEC-J2 (A) and HEK293 (B) cells were treated with different concentrations of the STAT3 inhibitor S3I-201 for 24 h, followed by infection with PEDV. At 48 hpi (IPEC-J2) or 24 hpi (HEK293), virus infection was determined by Western blotting with antibodies to PEDV N protein and β-actin (loading control). Densitometric data for the PEDV N protein/actin ratio from three independent experiments are expressed as means and SD. Virus RNA levels were also assessed by quantitative RT-PCR. (C and D) Knockdown of STAT3 expression reduced PEDV infection. Cells were transfected with STAT3-specific siRNA for 24 h and then infected with PEDV for 48 h or 24 h. STAT3 knockdown efficiency was verified by quantitative RT-PCR and Western blotting. The effect of STAT3 knockdown on virus loads was determined by Western blotting and quantitative RT-PCR. (E and F) Overexpression of STAT3 increased PEDV infection. IPEC-J2 (E) and HEK293 (F) cells were transfected with pAAV/STAT3 or vector control. Twenty-four hours later, the cells were incubated with PEDV for an additional 48 h or 24 h. The levels of STAT3 protein and PEDV N protein were analyzed by Western blotting. Total RNA was also extracted from the cells, and the viral RNA levels were then assessed by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Infection, Concentration Assay, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Over Expression, Plasmid Preparation, Incubation

    Depletion of EGFR expression enhanced the levels of several ISGs. IPEC-J2 (A) and HEK293 (B) cells were transfected with control siRNA or EGFR-specific siRNA for 24 h. Total RNA was extracted, and the mRNA levels of MxA , ISG15 , and IFN-β were determined by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: Depletion of EGFR expression enhanced the levels of several ISGs. IPEC-J2 (A) and HEK293 (B) cells were transfected with control siRNA or EGFR-specific siRNA for 24 h. Total RNA was extracted, and the mRNA levels of MxA , ISG15 , and IFN-β were determined by quantitative RT-PCR. The results are representative of three independent experiments (means and SD). *, P

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Expressing, Transfection, Quantitative RT-PCR

    EGFR inhibitors reduce PEDV infection. (A) Effects of inhibitors on EGFR function. IPEC-J2 and HEK293 cells were treated with the carrier control DMSO or EGFR-specific inhibitors, erlotinib (Erl) and gefitinib (Gef), at different concentrations for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. IPEC-J2 and HEK293 cells were also pretreated with an EGFR-specific inhibitor, Erl or Gef, at 10 μM or with DMSO for 12 h, followed by EGF stimulation (10 ng/ml) for 15 min. The levels of pEGFR and β-actin were analyzed by Western blotting. (B) EGFR inhibitors decrease the number of PEDV-positive cells. IPEC-J2 and HEK293 cells were pretreated with DMSO or EGFR inhibitors, Erl and Gef, at 10 μM for 12 h. After washing, the cells were infected with PEDV or mock control in the absence or presence of inhibitors. At 48 hpi or 24 hpi, the cell monolayers were fixed and examined for PEDV infection by IFA with an anti-PEDV spike protein MAb (3F12). The number of PEDV-positive cells was calculated. (C) Reduction of PEDV N protein by EGFR inhibitors is concentration dependent. Detergent lysates collected from IPEC-J2 and HEK293 cells were subjected to immunoblotting with antibodies as indicated. (D) EGFR inhibitors decreased PEDV RNA levels, as determined by quantitative RT-PCR. (E) Virus titers were reduced after inhibitor treatment, as detected by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: EGFR inhibitors reduce PEDV infection. (A) Effects of inhibitors on EGFR function. IPEC-J2 and HEK293 cells were treated with the carrier control DMSO or EGFR-specific inhibitors, erlotinib (Erl) and gefitinib (Gef), at different concentrations for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. IPEC-J2 and HEK293 cells were also pretreated with an EGFR-specific inhibitor, Erl or Gef, at 10 μM or with DMSO for 12 h, followed by EGF stimulation (10 ng/ml) for 15 min. The levels of pEGFR and β-actin were analyzed by Western blotting. (B) EGFR inhibitors decrease the number of PEDV-positive cells. IPEC-J2 and HEK293 cells were pretreated with DMSO or EGFR inhibitors, Erl and Gef, at 10 μM for 12 h. After washing, the cells were infected with PEDV or mock control in the absence or presence of inhibitors. At 48 hpi or 24 hpi, the cell monolayers were fixed and examined for PEDV infection by IFA with an anti-PEDV spike protein MAb (3F12). The number of PEDV-positive cells was calculated. (C) Reduction of PEDV N protein by EGFR inhibitors is concentration dependent. Detergent lysates collected from IPEC-J2 and HEK293 cells were subjected to immunoblotting with antibodies as indicated. (D) EGFR inhibitors decreased PEDV RNA levels, as determined by quantitative RT-PCR. (E) Virus titers were reduced after inhibitor treatment, as detected by TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Infection, CCK-8 Assay, Western Blot, Immunofluorescence, Concentration Assay, Quantitative RT-PCR

    (A to C) PEDV infection induces EGFR phosphorylation. IPEC-J2, HEK293, and Vero E6 cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed with PBS, and the cells were then cultured at 37°C for different times as indicated. Mock-infected cells were used as a control. Detergent lysates collected from the cells were directly subjected to reducing SDS-PAGE and blotted with MAbs against phospho-EGFR (pEGFR), EGFR, and β-actin. (D) UV-PEDV incubation activates EGFR. IPEC-J2 and HEK293 cells were incubated with UV-PEDV at an MOI of 1, and the cells were then further cultured for the indicated times. The cell lysates were subjected to immunoblotting with the antibodies indicated. (E) PEDV S-Flag protein interacts with EGFR. HEK293T cells were cotransfected with PEDV S-Flag (S) and EGFR (E). At 36 h after transfection, immunoprecipitation (IP) and immunoblotting were performed as described in Materials and Methods to examine interactions between PEDV S-Flag and EGFR.

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: (A to C) PEDV infection induces EGFR phosphorylation. IPEC-J2, HEK293, and Vero E6 cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed with PBS, and the cells were then cultured at 37°C for different times as indicated. Mock-infected cells were used as a control. Detergent lysates collected from the cells were directly subjected to reducing SDS-PAGE and blotted with MAbs against phospho-EGFR (pEGFR), EGFR, and β-actin. (D) UV-PEDV incubation activates EGFR. IPEC-J2 and HEK293 cells were incubated with UV-PEDV at an MOI of 1, and the cells were then further cultured for the indicated times. The cell lysates were subjected to immunoblotting with the antibodies indicated. (E) PEDV S-Flag protein interacts with EGFR. HEK293T cells were cotransfected with PEDV S-Flag (S) and EGFR (E). At 36 h after transfection, immunoprecipitation (IP) and immunoblotting were performed as described in Materials and Methods to examine interactions between PEDV S-Flag and EGFR.

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Infection, Incubation, Cell Culture, SDS Page, Transfection, Immunoprecipitation

    PEDV infection-induced STAT3 activation negatively regulates ISGs. (A and B) Infection with PEDV leads to STAT3 phosphorylation. IPEC-J2 (A) and HEK293 (B) cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed, and the cells were further incubated for different periods, as indicated. The cell lysates were blotted with the MAbs against phospho-STAT3 (pSTAT3), STAT3, and β-actin. The mock-infected cells were used as a control. (C and D) Inhibition of STAT3 function enhances antiviral responses. IPEC-J2 (C) and HEK293 (D) cells were treated with the STAT3-specific inhibitor S3I-201 (40 μM) for 24 h. The RNA levels of ISGs, MxA , ISG15 , and IFN-β , were determined by quantitative RT-PCR. (E) Effect of STAT3 inhibitor on cell viability. IPEC-J2 and HEK293 cells were treated with S3I-201 at the indicated concentrations or with the carrier control DMSO for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. The results are representative of three independent experiments (means and SD). *, P

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: PEDV infection-induced STAT3 activation negatively regulates ISGs. (A and B) Infection with PEDV leads to STAT3 phosphorylation. IPEC-J2 (A) and HEK293 (B) cells were incubated with PEDV at an MOI of 1 for 2 h at 4°C. Unbound viruses were removed, and the cells were further incubated for different periods, as indicated. The cell lysates were blotted with the MAbs against phospho-STAT3 (pSTAT3), STAT3, and β-actin. The mock-infected cells were used as a control. (C and D) Inhibition of STAT3 function enhances antiviral responses. IPEC-J2 (C) and HEK293 (D) cells were treated with the STAT3-specific inhibitor S3I-201 (40 μM) for 24 h. The RNA levels of ISGs, MxA , ISG15 , and IFN-β , were determined by quantitative RT-PCR. (E) Effect of STAT3 inhibitor on cell viability. IPEC-J2 and HEK293 cells were treated with S3I-201 at the indicated concentrations or with the carrier control DMSO for 72 h. Cell cytotoxicity was analyzed with the CCK-8 system as described in Materials and Methods. The results are representative of three independent experiments (means and SD). *, P

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Infection, Activation Assay, Incubation, Inhibition, Quantitative RT-PCR, CCK-8 Assay

    Knockdown of EGFR expression decreases PEDV infection. (A and C) Verification of EGFR knockdown efficiency. IPEC-J2 (A) and HEK293 (C) cells were transfected with EGFR-specific siRNA (siEGFR) or scrambled control siRNA (siCtrl) for 24 h, and the knockdown efficiency of EGFR was determined by quantitative RT-PCR and Western blotting. (B and D) Depletion of endogenous EGFR inhibited PEDV replication. After siRNA transfection for 24 h, cells were exposed to virus for 48 h (IPEC-J2) (B) or 24 h (HEK293) (D). The effect of EGFR knockdown on PEDV infection was determined by Western blotting (A and C, right), quantitative RT-PCR, and TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Journal: Journal of Virology

    Article Title: Porcine Epidemic Diarrhea Virus-Induced Epidermal Growth Factor Receptor Activation Impairs the Antiviral Activity of Type I Interferon

    doi: 10.1128/JVI.02095-17

    Figure Lengend Snippet: Knockdown of EGFR expression decreases PEDV infection. (A and C) Verification of EGFR knockdown efficiency. IPEC-J2 (A) and HEK293 (C) cells were transfected with EGFR-specific siRNA (siEGFR) or scrambled control siRNA (siCtrl) for 24 h, and the knockdown efficiency of EGFR was determined by quantitative RT-PCR and Western blotting. (B and D) Depletion of endogenous EGFR inhibited PEDV replication. After siRNA transfection for 24 h, cells were exposed to virus for 48 h (IPEC-J2) (B) or 24 h (HEK293) (D). The effect of EGFR knockdown on PEDV infection was determined by Western blotting (A and C, right), quantitative RT-PCR, and TCID 50 assay. The results are representative of three independent experiments (means and SD). *, P

    Article Snippet: IPEC-J2 cells (porcine small intestine epithelial cell clone J2; ATCC) and HEK293 cells (human embryonic kidney epithelial cells; ATCC) were cultured in Dulbecco's minimum essential medium (DMEM) (Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C in a 5% CO2 incubator (Thermo Scientific, USA).

    Techniques: Expressing, Infection, Transfection, Quantitative RT-PCR, Western Blot

    Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P

    Journal: Brazilian Journal of Medical and Biological Research

    Article Title: Oridonin induces growth inhibition and apoptosis in human gastric carcinoma cells by enhancement of p53 expression and function

    doi: 10.1590/1414-431X20187599

    Figure Lengend Snippet: Oridonin inhibited the growth, migration, and survivability of SNU-216 cells. Cells were treated with a series of oridonin (0, 10, 40 and 80 μM) for indicated days. A , Viable cell number was measured by trypan blue dye staining. B , Cell migration was assessed by wound healing assay. After treatment with oridonin for 24 h, the survival fraction of cells was detected by clonogenic assay ( C ), and cell viability of SNU-216 cells and HEK293 cells was measured by CCK-8 assay ( D ). Data are reported as means±SD. *P

    Article Snippet: The human GC cell line SNU-216 and human kidney epithelial cell line HEK293 were purchased from the American Type Culture Collection (ATCC, USA).

    Techniques: Migration, Staining, Wound Healing Assay, Clonogenic Assay, CCK-8 Assay