Journal: Biomacromolecules

Article Title: Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

doi: 10.1021/acs.biomac.1c01280

Figure Lengend Snippet: Impact of engineered GFPp_FGF1 oligomers on FGFR1 binding and activation. (A) Serum-starved NIH3T3 cells were treated with increasing concentrations of the wild-type FGF1 or GFPp_FGF1 oligomers. Cells were lysed, and activation of FGFR1 and receptor-downstream signaling were assessed with Western blotting. The level of tubulin served as a loading control. (B) To determine the kinetics of FGFR1 signaling upon cell stimulation with GFPp_FGF1, serum-starved NIH3T3 cells were stimulated with proteins for up to 6 h. At distinct time points of incubation, cells were lysed and analyzed by Western blotting. (C) Kinetics of the interaction of GFPp_FGF1 oligomers with FGFR1 was analyzed using biolayer interferometry (BLI). The extracellular region of FGFR1 (FGFR1ecd-Fc) was immobilized on Protein A sensors, and then, the receptor was incubated with distinct GFPp_FGF1 oligomers. The association and dissociation profiles were measured.

Article Snippet: Mouse embryo fibroblast cells (NIH3T3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Binding Assay, Activation Assay, Western Blot, Control, Cell Stimulation, Incubation

Journal: Biomacromolecules

Article Title: Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

doi: 10.1021/acs.biomac.1c01280

Figure Lengend Snippet: Engineering of the fluorescent trimeric cytotoxic conjugate targeting FGFR1. (A) The C-terminal LPETGG sequence was incorporated into the trimeric GFPp_FGF1E via gene synthesis, yielding 3xGFPp_FGF1E_LPETGG. Sortase A recognizes the LPETGG sequence within 3xGFPp_FGF1E_LPETGG and mediates ligation of the tetraglycine peptide-linked MMAE to 3xGFPp_FGF1E_LPETGG, resulting in 3xGFPp_FGF1E_LPET_MMAE. (B) The efficiency of the conjugation and purity of the obtained 3xGFPp_FGF1E_LPET_MMAE were confirmed by SDS-PAGE. (C) The site-specific attachment of MMAE to 3xGFPp_FGF1E_LPETGG was confirmed by MALDI-MS. The impurities that appear in MALDI-MS (about 30,000 Da) are either the result of a minor protein fragmentation during ionization or trace impurities/degradation products not visible in SDS-PAGE and UV spectra but detectable in the high-sensitivity MS approach. (D) Assessment of the biological activity of recombinant proteins. Serum-starved NIH3T3 cells were incubated with FGF1 WT (positive control) or with 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE. Cells were lysed, and activation of FGFR1 was assessed with Western blotting. The level of tubulin served as a loading control. (E) Binding profiles of 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE to FGFR1 were measured using BLI. The extracellular region of FGFR1 (FGFR1ecd-Fc) was immobilized on Protein A sensors and incubated with proteins/conjugates. Association and dissociation profiles were measured.

Article Snippet: Mouse embryo fibroblast cells (NIH3T3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Sequencing, Ligation, Conjugation Assay, SDS Page, Activity Assay, Recombinant, Incubation, Positive Control, Activation Assay, Western Blot, Control, Binding Assay

Journal: Biomacromolecules

Article Title: Intrinsically Fluorescent Oligomeric Cytotoxic Conjugates Toxic for FGFR1-Overproducing Cancers

doi: 10.1021/acs.biomac.1c01280

Figure Lengend Snippet: Stability analysis of the 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE. (A) 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE were incubated in human serum in the presence of heparin at 37 °C for 96 h. At distinct time points (0, 24, 48, 72, and 96 h), samples were taken, and the oligomeric state of proteins was analyzed using native PAGE UV light imaging. (B) The stability of the GFPp oligomerization scaffold within the trimeric protein and the conjugate was determined by monitoring GFP fluorescence at distinct time points of incubation in human serum at 37 °C. Fluorescence spectra were acquired using a FP-8500 spectrofluorometer (Jasco, Japan) with excitation at 488 nm and emission in the 500–650 nm range. (C) The stability of FGF1E in 3xGFPp_FGF1E_LPETGG and 3xGFPp_FGF1E_LPET_MMAE was determined with Western blotting using antibodies recognizing FGF1. (D) Evaluation of the biological activity of 3xGFPp_FGF1E_LPETGG and its cytotoxic conjugate. Samples were incubated with human serum at 37 °C for 96 h. At distinct time points of incubation (0, 24, 48, 72, and 96 h), proteins were added to serum-starved NIH3T3 cells. Cells were lysed, and activation of FGFR1 and receptor-downstream signaling were assessed with Western blotting. The level of tubulin served as a loading control.

Article Snippet: Mouse embryo fibroblast cells (NIH3T3) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Clear Native PAGE, Imaging, Fluorescence, Western Blot, Activity Assay, Activation Assay, Control

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Isolation of prelatent antithrombin by heparin-agarose chromatography. Left, elution profile of heat-treated native plasma antithrombin after chromatography on a Hi-Trap heparin column (solid line). Protein was eluted from the column with a linear NaCl gradient (dashed line) and was detected by intrinsic fluorescence. The protein peak eluting between 1 and 2 m NaCl was subdivided as shown into two pools, and the leading edge pool was rechromatographed and similarly subdivided to obtain the three pools A, B, and C (A–C), as described under “Experimental Procedures.” Right, elution profiles of antithrombin pools A, B, and C after rechromatography on the Hi-Trap Heparin column using a convex salt gradient (dashed line). Latent antithrombin and untreated native antithrombin were similarly chromatographed. All chromatograms were corrected for background fluorescence by subtracting a chromatogram of buffer alone. Further details are provided under “Experimental Procedures.”

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Isolation, Chromatography, Clinical Proteomics, Fluorescence

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Kinetics of conversion of native antithrombin to prelatent and latent forms. A, fluorescence elution profiles obtained after heating native plasma antithrombin at 60 °C in pH 7.4 citrate buffer for the indicated times (in h) and chromatographing 10 μg samples on the Hi-Trap heparin column as in Fig. 1 using the convex salt gradient (dashed line). The different forms of antithrombin eluting from the column were quantitated by integrating the areas under each peak, and the relative amounts of each form were expressed as a percentage of the total fluorescence. Further details are provided under “Experimental Procedures.” B, plot of the time dependence for conversion of native antithrombin (AT) (•) to prelatent (▪), latent (▴), and nonbinding (♦) forms based on the quantitation of these forms in the chromatograms of A and others not shown for clarity. The solid lines indicate the computer fit of data by the model in D along with the fitted rate constants as detailed under “Experimental Procedures.” C, chromatograms of isolated prelatent antithrombin after incubating at 60 °C in pH 7.4 Tris/citrate buffer for the indicated times (in h) and chromatographing on Hi-Trap Heparin with elution using the convex salt gradient (dashed line). Prelatent, latent, and unbound forms of antithrombin were quantitated by integration of peaks and normalizing to the total fluorescence as in A. D, minimal kinetic model consistent with the data of A–C together with the rate constants providing the best fit of the data by this model.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Fluorescence, Clinical Proteomics, Quantitation Assay, Isolation

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Binding of pentasaccharide (H5) and full-length (H26) high affinity heparins to native and prelatent antithrombins Native and prelatent antithrombins were titrated with the indicated high affinity heparins under stoichiometric binding conditions ( I 0.05) or under conditions where K D was well determined ( I 0.15 or I 0.3) as indicated below and in . Binding was monitored from increases in tryptophan fluorescence as in previous studies, and binding parameters were determined by fitting titration curves by the equilibrium binding equation, as described under “Experimental Procedures.” Binding stoichiometries determined in I 0.05 buffer were fixed in fits of titrations in I 0.15 or I 0.3 buffers. Only an upper limit for K D in I 0.05 buffer is provided, because binding was too tight to measure accurately in these titrations. Errors represent ± S.E. for 3–7 titrations.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Binding Assay, Fluorescence, Titration

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Comparison of the kinetics and affinity of the interaction of heparin with native and prelatent antithrombins. A and B compare representative fluorescence titrations of native (•) and prelatent (○) forms of antithrombin (AT) with the heparin pentasaccharide performed under stoichiometric binding conditions (I 0. 05) (A) or under equilibrium binding conditions (I 0.15) (B) as described under “Experimental Procedures.” Titrations were fit by the quadratic binding equation (solid lines) to obtain values for the binding stoichiometry, KD, and the maximal fluorescence change (Table 2). The fitted stoichiometry for the titration at I 0.05 was fixed in fitting the titration at I 0.15. C compares the kinetics of pentasaccharide binding to native (•) and prelatent (○) antithrombins under pseudo-first order conditions as a function of the pentasaccharide concentration. Solid lines are linear regression fits of data from which kon and koff were determined from the slope and intercepts, respectively.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Comparison, Fluorescence, Binding Assay, Titration, Concentration Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: SDS and native PAGE characterization of prelatent antithrombin. Shown are the electrophoretic gels of antithrombin pools A, B, and C (5 μg of protein) obtained during the purification of prelatent antithrombin by Hi-Trap heparin chromatography (Fig. 1) under denaturing (SDS) and native conditions. Native and latent antithrombin samples were run as controls. The ability of each antithrombin (AT) pool to form an SDS-stable complex with a molar excess of thrombin (5 μg) is shown in the SDS gel.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Clear Native PAGE, Purification, Chromatography, SDS-Gel

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Stoichiometries and kinetics of inhibition of thrombin and factor Xa by native and prelatent antithrombins Untreated antithrombin (AT) and the different pools obtained from Hi-Trap heparin chromatography of heat-treated antithrombin were compared with respect to (i) the stoichiometries of inhibition (SI) of thrombin (IIa) and (ii) the second order rate constants for the inhibition of thrombin or factor Xa (FXa) in the absence of heparin ( k uncat ) or for the inhibition of factor Xa in the presence of saturating heparin pentasaccharide ( k H5 ) as described under “Experimental Procedures.” Errors represent S.E. obtained from the fits of stoichiometric titrations or reaction kinetic curves as a function of time or heparin concentration.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Inhibition, Chromatography, Concentration Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Antiproliferative activity of prelatent antithrombin. HUVECs were cultured with or without stimulation by FGF-2 and in the absence or presence of prelatent (pool A), latent, and native (untreated and pool C) forms of antithrombin (AT) (see Fig. 1) as indicated for 48 h. The number of viable cells was then assayed colorimetrically. Triplicate assays were performed for each condition, and results were expressed relative to the unstimulated control. Further details are provided under “Experimental Procedures.” Error bars, S.E. values obtained after grouping results from several independent experiments. Prelatent and latent antithrombins produced statistically significant growth inhibition (p < 0.01) at minimal doses of 10 and 50 μg/ml, respectively.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Activity Assay, Cell Culture, Control, Produced, Inhibition

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Comparison of high affinity heparin-catalyzed reactions of native and prelatent antithrombin with proteases. Native (•) or prelatent (○) antithrombins (∼20 nm) were reacted with 1 nm thrombin (left) or 2 nm factor Xa (right) in the presence of variable concentrations of high affinity heparin (HA-heparin) for fixed times of 2 min (thrombin) or 30 s (factor Xa). Thrombin reactions additionally contained 2 mm p-aminobenzamidine to slow the rate sufficiently to allow accurate kinetic measurements. Apparent second order rate constants (kapp) were obtained by dividing observed pseudo-first order rate constants for protease inactivation by the functional antithrombin concentration. Additional corrections were made for the competitive effect of p-aminobenzamidine in reactions with thrombin. The bell-shaped heparin concentration dependence of kapp was fit by the ternary complex model (solid lines for native antithrombin, dashed lines for prelatent antithrombin) to provide values for the binary protein-heparin complex dissociation constants and the true second order rate constants for the reactions of antithrombin-heparin binary complex with each protease (32). These values are tabulated in Table 3. Further details are provided under “Experimental Procedures.”

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Comparison, Functional Assay, Concentration Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Kinetics of heparin-catalyzed reactions of native and prelatent antithrombins with thrombin and factor Xa Kinetic parameters were obtained from fits of the data of Figs. ​ Figs.6 6 and for high affinity heparin and low affinity heparin-catalyzed reactions of native and prelatent antithrombins with thrombin or factor Xa by the ternary complex bridging or conformational activation models described under “Experimental Procedures.” The fitted parameters were the rate constant for the reaction of heparin-complexed antithrombin with protease ( k H ) and the dissociation constants for the binary antithrombin-heparin complex ( K AT,H ) and the protease-heparin binary complex ( K Pr,H ).

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Activation Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Comparison of low affinity heparin-catalyzed reactions of native and prelatent antithrombins with proteases. Native (•) and prelatent (○) antithrombins were reacted with thrombin (20 nm inhibitor and 1 nm protease) or with factor Xa (50 nm inhibitor and 5 nm protease) in the presence of increasing concentrations of low affinity heparin (LA-heparin) for fixed times of 5 min or for variable reaction times. Apparent second order inactivation rate constants were calculated from observed pseudo-first order rate constants and the functional inhibitor concentration as in Fig. 6. The heparin concentration dependence of kapp was fit by the ternary complex bridging model for reactions with thrombin or by a model in which conformational activation of antithrombin solely contributed to the rate-enhancing effect of heparin for reactions with factor Xa (32). Solid lines indicate the fit of the native antithrombin kinetic data, and dashed lines indicate the fit of the prelatent antithrombin kinetic data.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Comparison, Functional Assay, Concentration Assay, Activation Assay

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Decreased thermal stability of prelatent antithrombin. Shown are melting curves for native (•), prelatent (○), and latent (▵) forms of antithrombin measured from decreases in intrinsic protein fluorescence as a function of increasing temperature (in degrees Kelvin). Data were fit by the van't Hoff equation for a two-state unfolding transition to obtain the melting temperature corresponding to the midpoint of the unfolding curves, as described under “Experimental Procedures.” Fitted melting temperatures are reported in degrees centigrade. The results are representative of three independent experiments.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Fluorescence

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Analysis of conformational alterations in prelatent antithrombin by proteolytic susceptibility. The susceptibility of native, prelatent, and latent forms of antithrombin to digestion by catalytic levels of the nontarget proteases, chymotrypsin and thermolysin (serpin/protease weight ratio of 10:1), was monitored as a function of digestion time by SDS-PAGE as described under “Experimental Procedures.”

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: SDS Page

Journal:

Article Title: Characterization of the Conformational Alterations, Reduced Anticoagulant Activity, and Enhanced Antiangiogenic Activity of Prelatent Antithrombin *

doi: 10.1074/jbc.M710327200

Figure Lengend Snippet: Conformational alterations in prelatent antithrombin probed by 1H NMR spectroscopy. Comparison of 900 MHz 1H NMR spectra of 10 μm samples of native (N), prelatent (P), and latent (L) antithrombins in I 0.15, pH 7.4, D2O buffer at 25 °C. Samples were prepared, and spectra were recorded as described under “Experimental Procedures.” Difference spectra between native and prelatent, native and latent, and prelatent and latent are shown to emphasize the similarity of native and prelatent spectra and the significant differences between either of these forms and latent antithrombin. The spike at 3.7 ppm represents small differences in the H2O content of the samples.

Article Snippet: Reactive loop-cleaved antithrombin was prepared by incubating native antithrombin with human neutrophil elastase (Athens Research Technology) as in previous studies ( 31 , 32 ).

Techniques: Structural Proteomics, Comparison

Journal: PLoS Pathogens

Article Title: Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense

doi: 10.1371/journal.ppat.1006713

Figure Lengend Snippet: (A) Immunoblotting of phosphorylated-IRF3 (p-IRF3), SeV, EAP30 and action levels in PH5CH8 cells transfected with control or EAP30 siRNA for 48 h and mock-infected or infected with SeV for additional 8 h. (B) Immunoblotting of IRF3 monomer and dimer forms following native PAGE of the samples shown in (A). (C) Immunoblotting of IRF3, SeV, EAP30, lamin A/C (nuclear protein marker), β-tubulin (cytoplasmic protein marker), and actin loading control in cytoplasmic (CE) and nuclear (NE) fractions of PH5CH8 cells transfected with control or EAP30 siRNA and mock-infected or infected with SeV. (D) Immunoblotting of p-IRF3, actin, ISG56, SeV, and EAP30 in HEK293-shEAP30 and HEK293-shCon cells mock-infected or infected with SeV for 8 h. (E) Whole cells lysate (WCL) were collected from HEK293-shEAP30 and HEK293-shCon cells that were mock-infected or infected with SeV for immunoblotting of CBP, IRF3, EAP30 and actin (left panel) and co-IP analysis of virus-induced CBP-IRF3 association (right panel). (F) ChIP analysis of IRF3 binding to IFNβ, IFNL1, and IFIT1 (ISG56) promoters in nuclear extracts of HEK293-shEAP30 and HEK293-shCon cells that were mock-infected or infected with SeV. The ChIP-enriched DNA levels were analyzed by qPCR and normalized to input DNA, followed by subtraction of nonspecific binding determined using control IgG. “*” denotes statistical differences exist with a P -value of < 0.05.

Article Snippet: PH5CH8 non-neoplastic hepatocytes (provided by Nobuyuki Kato, Okayama University, Japan) [ ], human hepatoma Huh7.5-TLR3 and Huh7-TLR3 cells that were stably reconstituted for the expression of human TLR3 (developed in this laboratory) [ , ], and human embryo kidney HEK293 cells (obtained from American Type Culture Collection), were maintained as described previously [ , , ].

Techniques: Western Blot, Transfection, Control, Infection, Clear Native PAGE, Marker, Co-Immunoprecipitation Assay, Virus, Binding Assay

Journal: PLoS Pathogens

Article Title: Pivotal role for the ESCRT-II complex subunit EAP30/SNF8 in IRF3-dependent innate antiviral defense

doi: 10.1371/journal.ppat.1006713

Figure Lengend Snippet: (A) HEK293 (5x10 4 ) cells were transfected with 100 ng of each plasmid or various double/triple plasmid combinations as indicated, with control vector being added to keep the total amount (300 ng) of transfected DNA constant in each condition. 48 h later, cells were challenged with VSV-Luc (MOI = 0.1) for 6 h (except the mock group) followed by cell lysis and luciferase assay. (B) Immunoblot analysis of transfected EAP30, EAP20 (using anti-HA), CBP, IRF3 (using anti-FLAG) and endogenous actin under experimental conditions of (A) for each transfection groups. (C-E) qPCR analysis of IFN-β (C), OASL (D), and IFN-λ1 (E) mRNA levels in HEK293 cells transfected with different plasmid combinations similar to panel (A) for 48 h. “*”, “**”, and “***” denote statistical differences exist as compared with empty vector-transfected cells with a P -value of < 0.05, < 0.01, and <0.001, respectively.

Article Snippet: PH5CH8 non-neoplastic hepatocytes (provided by Nobuyuki Kato, Okayama University, Japan) [ ], human hepatoma Huh7.5-TLR3 and Huh7-TLR3 cells that were stably reconstituted for the expression of human TLR3 (developed in this laboratory) [ , ], and human embryo kidney HEK293 cells (obtained from American Type Culture Collection), were maintained as described previously [ , , ].

Techniques: Transfection, Plasmid Preparation, Control, Lysis, Luciferase, Western Blot