Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Dimerization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Nuclear translocation of IRF3 in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). GAPDH and LaminA/C were used as cytoplasm and nuclear protein loading controls. ( D ) Immunofluorescence images showing the impact of 14,15-EET and TPPU on the subcellular localization of IRF3 in uninfected (CON) or CVB3-infected AC16 cells; bars =50 μm. ( E ) Scheme showing the two main RNA sensor-induced IRF3 activation pathways. ( F-G ) AC16 cells that were uninfected (CON) or infected with CVB3 were treated with 11,12-EET, 14,15-EET or TPPU (n=3). Shown are changes in IRF3 phosphorylation following the siRNA-mediated down regulation of MDA5 (F), or TLR3 (G). ( H ) Activity of a IFNβ-luciferase reporter construct in HEK293 cells expressing either a control vector (vector), pcMDA5, pcMAVS, pcTBK1 , or pcIRF3-5D and treated with solvent (CON) or 14,15-EET (1 μM) for 24 hours. ( I ) Impact of the siRNA-mediated downregulation of TBK1 on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Translocation Assay, Immunofluorescence, Activation Assay, Activity Assay, Luciferase, Construct, Expressing, Control, Plasmid Preparation, Solvent

Journal: bioRxiv

Article Title: Epoxyeicosatrienoic acids and sEH inhibition prevent cardiac dysfunction in CVB3-induced myocarditis by positively regulating type I interferon signaling

doi: 10.1101/2023.02.03.527086

Figure Lengend Snippet: ( A ) Impact of the siRNA-mediated downregulationof GSK3β on the phosphorylation of IRF3 (on Ser396) in uninfected (CON) or CVB3-infected AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( B ) Impact of the EET antagonist; EEZE (1 μM) on the phosphorylation of GSK3β in AC16 cells treated with 11,12-EET, 14,15-EET or TPPU (n=4). ( C ) Co-immunoprecipitation of TBK1 with a GSK3β-Flag fusion protein from HEK293 cells treated with 14,15-EET (1 μM) or TPPU (10 μM) in the absence or presence of EEZE (n=3). ( D ) Impact of the wild-type GSK3β and the Y216F and Y216D GSK3β mutants on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3). ( E-F ) Impact of GSK3β mutation alone and in combination with 14,15-EET on the phosphorylation of TBK1 in uninfected and CVB3-infected HEK293 cells (n=3-4). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: After 48 hours, transfected HEK293 cells were harvested in immunoprecipitation (IP) buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 1% Triton X-100 with freshly added PMSF, protease inhibitors and phosphatase inhibitors (Boster Biological Tech, Wuhan, China).

Techniques: Phospho-proteomics, Infection, Immunoprecipitation, Mutagenesis

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A Immunofluorescence analysis of PD-1 binding to PD-L1 on RKO cells treated with TER. RKO cells were incubated with recombinant human PD-1 Alexa 647 protein and treated with the indicated concentrations of TER. Red fluorescence represents PD-1 binding; the nuclei were stained with Hoechst (blue). B Competitive ELISA assay measuring the inhibition of PD-1/PD-L1 interaction by TER. The binding between PD-1 and PD-L1 in the presence of increasing concentrations of TER was assessed. C The 3D structure of TER (upper panel) and its predicted binding pose within the active site of PD-1 (PDB ID: 3RRQ, lower panel). The key interacting residues include PHE56, SER57, ASN58, THR59, SER62, PHE63, PHE82, PRO83, GLN99, ASN102, GLY103, and ARG104. D Protein–ligand complex showing TER docked to PD-1, with the binding pocket highlighted. E Surface plasmon resonance (SPR) analysis showing the binding of TER to PD-1. αPD-L1 was used as a positive control. *** p < 0.001 and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant Jurkat-T cells expressing human PD-1 and an NFAT reporter gene (hPD-1/NFAT Jurkat-T cells, #60535) and recombinant CHO-K1 cells expressing human PD-L1 and a T-cell receptor (TCR) activator (hPD-L1/TCR CHO-K1 cells, #60536) were obtained from BPS Bioscience.

Techniques: Immunofluorescence, Binding Assay, Incubation, Recombinant, Fluorescence, Staining, Competitive ELISA, Inhibition, SPR Assay, Positive Control, Control

Journal: Oncogenesis

Article Title: Teriflunomide modulates the PD-1/PD-L1 axis and enhances antitumor immunity in colorectal cancer

doi: 10.1038/s41389-026-00607-3

Figure Lengend Snippet: A The viability of hPD-1 Jurkat-T cells and hPD-L1 CHO cells following treatment with the indicated concentrations of TER for 24 h. B Luciferase activity measured using a PD-1/PD-L1 blockade bioassay. hPD-1 Jurkat-T cells (effector cells) were co-cultured with hPD-L1-expressing aAPC/CHO-K1 cells (target cells) in the presence of indicated concentrations of TER. The luminescence signal indicates the level of TCR signaling activation. αPD-L1 was used as a positive control. * <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the respective control.

Article Snippet: Recombinant Jurkat-T cells expressing human PD-1 and an NFAT reporter gene (hPD-1/NFAT Jurkat-T cells, #60535) and recombinant CHO-K1 cells expressing human PD-L1 and a T-cell receptor (TCR) activator (hPD-L1/TCR CHO-K1 cells, #60536) were obtained from BPS Bioscience.

Techniques: Luciferase, Activity Assay, Bioassay, Cell Culture, Expressing, Activation Assay, Positive Control, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of small molecules and a druggable groove that regulate DNA binding and release of the AP-1 transcription factor ΔFOSB

doi: 10.1016/j.jbc.2025.111080

Figure Lengend Snippet: Cellular activity of JPC0661 . A , cell viability of JPC0661 comm and HTS10307 assessed in the range of 0 to 20 μM after 24 h ( top ) and 72 h ( bottom ) in neural progenitor cells (NPCs) using the CellTiter-Glo viability assay. Cell viability measures are normalized to the negative control containing no compound but 0.6% DMSO. Dotted lines indicate 100% cell viability. The mean of n replicates is shown for 24 h (n = 3) and 72 h (n = 2) with error bars indicating the SEM. B , stability of JPC0661 comm and HTS10307 in mouse liver microsomes. C and D , effects of JPC0661 comm (0–100 μM; ( C ) and JPC0661 (0–100 μM; ( D ) on AP-1-driven luciferase activity in AP-1-luc HEK293 cells. The dose-dependent activation of the AP-1-driven luciferase reporter was measured based on changes in the luciferase signal and expressed as relative fluorescence units (RFUs). Each compound was tested twice (n = 4 wells per experiment; total n = 6–8 wells for JPC0661 comm and n = 7–8 wells for JPC0661 per concentration), with results normalized to the luciferase signals of blank wells from corresponding experiments (n = 8 wells). Nonlinear regression using a three-parameter model was applied to fit the luciferase signal and calculate IC 50 values, which are reported with a 95% confidence interval (CI), and data points are presented as the mean ± SEM. E , effect of serially diluted JPC0661 (0.003–100 μM) on the viability of AP-1-luc HEK293 cells using the Celltiter-Glo viability assay (2 h). Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 7 wells). Data points are presented as the mean ± SEM, with a total n = 10 to 12 wells per concentration. F , effect of JPC0661 (at doses 12.5, 25, and 50 μM) on the viability of Neuro 2A cells (72 h) using the Celltiter-Glo viability assay to assess potential toxicity in mouse neuronal cells. Cell viability was normalized to the DMSO control, which contained 0.5% DMSO but no compound (n = 8 wells). Data points are presented as the mean ± SEM, with a total n = 12 wells per concentration. AP-1, activator protein 1; DMSO, dimethyl sulfoxide.

Article Snippet: The AP-1-luciferase reporter HEK293 cell line was obtained from BPS Bioscience and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Activity Assay, Viability Assay, Negative Control, Luciferase, Activation Assay, Fluorescence, Concentration Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Discovery of small molecules and a druggable groove that regulate DNA binding and release of the AP-1 transcription factor ΔFOSB

doi: 10.1016/j.jbc.2025.111080

Figure Lengend Snippet: Validation of JPC0661 as an inhibitor of ΔFOSB . A , a panel of JPC0661 analogs was designed to probe the role of the sulfonic acid group and the amino-pyrazolone group. B , the table summarizing the JPC0661 analogs tested in AP-1-reporter assays using AP-1-luc HEK293 cells, yielding cell-based IC 50 values and FP-DRC assays. Lower IC 50 values are marked with more plus signs (+) and indicate higher activity. The plots from the FP-DRC and cell-based DRC assays for these compounds are shown in . C , ΔFOSB–JUND bZIP incubated with compounds (0.5 mM) with 100 μM diamide (“ ox ,” oxidized) or without diamide (“ red ,” reduced) and assessed by SDS-PAGE (with or without reducing agent in the loading buffer). D , ΔFOSB–JUND bZIP protein incubated with compounds (0.5 mM) with no diamide (“ red ”) or protein alone (no compound) incubated with diamide (“ ox ”) as a control and then assessed by SDS-PAGE (with or without reducing agent in the loading buffer). In ( C ) and ( D ), “cntrl” denotes the ΔFOSB–JUND bZIP protein in the absence of compound. AP-1, activator protein 1; bZIP, basic leucine zipper; FP-DRC, fluorescence polarization–dose–response curve; HEK293, human embryonic kidney 293 cell line.

Article Snippet: The AP-1-luciferase reporter HEK293 cell line was obtained from BPS Bioscience and maintained in growth medium 1B (BPS Bioscience) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin–streptomycin (Hyclone) under standard incubation conditions of 37 °C with 5% CO 2 .

Techniques: Biomarker Discovery, Activity Assay, Incubation, SDS Page, Control, Fluorescence

Journal: Chemical biology & drug design

Article Title: 2-Aminothiophene derivatives as a new class of positive allosteric modulators of glucagon-like peptide 1 receptor

doi: 10.1111/cbdd.14039

Figure Lengend Snippet: Potential allosteric effect of S-1 and S-2 on human GLP-1R. (a) The effect of GLP-1 on HEK293 cells expressing human GLP-1R or empty vector in the presence or absence of S-1 (23.5 μM). (b) The effect of GLP-1 on HEK293 cells expressing human GLP-1R or empty vector in the presence or absence of S-2 (71 μM). GLP-1R activation was assessed as luminescence normalized to protein concentration and plotted as luminescence fold change with respect to vehicle control (0.5% DMSO). Data are average of three independent experiments with at least three technical replicates for each conditions, and error bars for each concentration were plotted as SEM (n = 3). Statistical analysis was done using 2-way ANOVA (****p < 0.0001; **p < 0.001; *p < 0.5). The comparison is done between the data points of the dose–response curve generated due to GLP-1’s effect on GLP-1R in the presence and absence of S-1 or S-2

Article Snippet: HEK293 cell stably expressing CRE/CREB luciferase reporter gene (BPS Bioscience #60515), RPMI medium (Corning #10-040), Krebs-Ringer Solution, HEPES-buffered (Alfa Aesar #J67795-K2), L-Glutamine (Gibco #25030-081), HEPES (Gibco #15630-080), Sodium Pyruvate (Gibco #11360-070), β-mercaptoethanol (MP #806444), D-Glucose (#G-7528), Fetal Bovine Serum (Fisher #03600511), penicillin/streptomycin (Corning #30-002-Cl), Hygromycin B (Alfa Aesar #J60681), Lipofectamine 2000 (Invitrogen #11668027), vasoactive intestinal peptide receptor (VIPR) peptide agonist (Sigma #V3628), VIPR peptide antagonist (Sigma #SCP0260), GLP-1R peptide agonist (Sigma #9416), 6-well cell culture plates (Ultra Cruz #sc-204443), 96-well cell culture plates (Sigma #CLS9102), Luciferase cell culture lysis reagent (Promega #E1531), Luciferase assay reagent (Promega #E1501), and Ultra-Sensitive Rat Insulin Kit (Crystal Chem #90060) were purchased from vendors.

Techniques: Expressing, Plasmid Preparation, Activation Assay, Protein Concentration, Control, Concentration Assay, Comparison, Generated