Journal: Food & function

Article Title: Macrophage activation by edible mushrooms is due to the collaborative interaction of toll-like receptor agonists and dectin-1b activating beta glucans derived from colonizing microorganisms

doi: 10.1039/c9fo01707k

Figure Lengend Snippet: Mushroom macrophage stimulatory activity exhibited by water-soluble extracts is predominantly due to TLR agonists. Extracts from 13 types of mushrooms were evaluated in HEK-Blue™ hTLR2 cells for TLR2-dependent activity (A), HEK-Blue™ hTLR4 cells for TLR4-dependent activity (B), and control HEK-Blue™ null1 cells (C). Response ratio is defined as OD of sample/OD of negative control. Positive controls ultra pure E. coli LPS and Pam3CSK4 were each tested at 100ng/ml. TLR2- and TLR4-dependent activities exhibited by extracts of the 13 types of mushrooms (n=26) were plotted against TNF-α production from RAW 264.7 macrophages (D and E, respectively). EC25 values represent the concentration (μg/ml) of mushroom material required to induce cytokine production to levels 25% of those achieved by ultra pure E. coli LPS (100 ng/ml).

Article Snippet: Selective detection of activation of the various pathogen recognition receptors (PRR) was accomplished using HEK-BlueTM hTLR2, HEK-BlueTM hTLR4, HEK-BlueTM hDectin-1a, HEK-BlueTM hDectin-1b and HEK-BlueTM Null1 cells (InvivoGen).

Techniques: Activity Assay, Control, Negative Control, Concentration Assay

Journal: Food & function

Article Title: Macrophage activation by edible mushrooms is due to the collaborative interaction of toll-like receptor agonists and dectin-1b activating beta glucans derived from colonizing microorganisms

doi: 10.1039/c9fo01707k

Figure Lengend Snippet: Collaborative interaction between TLR agonists and dectin-1b agonists within shiitake (Lentinula edodes). A soluble extract was prepared containing only TLR2 and TLR4 agonists (A) and an insoluble extract was prepared containing water-insoluble particulate matter exhibiting only dectin-1b dependent activity (B). Presence/absence of activity was determined using ANOVA followed by pairwise t-test. Selective detection of the pathogen recognition receptors was accomplished using HEK-Blue™ hdectin-1b cells, HEK-Blue™ hTLR2 cells, HEK-Blue™ hTLR4 cells, and control HEK-Blue™ null1 cells (A, B). RAW 264.7 macrophages were treated with the soluble (black squares) and insoluble extracts (black circles) and in combination (white circles) at the indicated concentrations (C). Theoretical values for an additive interaction between the soluble and insoluble extracts is indicated by the dashed line with “X” symbols. Macrophages were treated for 18 hours and TNF-α in culture supernatant was measured by ELISA. Treatment with “Soluble + Insoluble Exts” significantly enhanced TNF-α production compared to other treatments, including the “Theoretical additive value”. *p<0.05, **p<0.001.

Article Snippet: Selective detection of activation of the various pathogen recognition receptors (PRR) was accomplished using HEK-BlueTM hTLR2, HEK-BlueTM hTLR4, HEK-BlueTM hDectin-1a, HEK-BlueTM hDectin-1b and HEK-BlueTM Null1 cells (InvivoGen).

Techniques: Activity Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Cytotoxicity of dioncophylline A and related naphthylisoquinolines in leukemia cells, mediated by NF-κB inhibition, angiogenesis suppression, G2/M cell cycle arrest, and autophagy induction.

doi: 10.1016/j.phymed.2023.155267

Figure Lengend Snippet: Fig. 1. Cytotoxic effect of naphthylisoquinolines, Cpds. 1–4, by the resazurin assay. (A) Dose-response curves of dioncophylline A and its derivatives, with the positive control doxorubicin on CCRF-CEM and CEM-ADR5000 cells. (B) Cytoxicity of dioncophylline A on normal healthy cells (PBMC) and HEK-Blue™ Null1 cells upon dioncophylline A treatment. The data are the result of mean values ± SD of three independent experiments.

Article Snippet: The NF-κB SEAP reporter parental cell line HEK-BlueTM Null1 was purchased from InvivoGen (San Diego, CA, USA) and cultured under the recommended conditions.

Techniques: Resazurin Assay, Positive Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Cytotoxicity of dioncophylline A and related naphthylisoquinolines in leukemia cells, mediated by NF-κB inhibition, angiogenesis suppression, G2/M cell cycle arrest, and autophagy induction.

doi: 10.1016/j.phymed.2023.155267

Figure Lengend Snippet: Fig. 9. Immunofluorescence visualizes the NF-κB translocation in HEK-Blue™ Null1 cells. TNF-α enhanced NF-κB translocation from the cytoplasm to the nucleus in the absence of dioncophylline A. However, TNF-α induced NK-κB translocation was inhibited by pre-treatment with dioncophylline A (1, 5, and 10 µM) for 24 h. An AF7000 widefield fluorescence microscope at 40 × magnification (scale bars = 10 µm) was used for visualization.

Article Snippet: The NF-κB SEAP reporter parental cell line HEK-BlueTM Null1 was purchased from InvivoGen (San Diego, CA, USA) and cultured under the recommended conditions.

Techniques: Immunofluorescence, Translocation Assay, Fluorescence, Microscopy

Journal: Advanced Biomedical Research

Article Title: Immunomodulatory Activity of Polysaccharide from Trametes gibbosa (Pers.) Fr (Basidiomycota, Fungi) Mediated by TLR4 Signaling Pathway

doi: 10.4103/abr.abr_50_22

Figure Lengend Snippet: Effects of T. gibbosa polysaccharide fraction on cell viability of HEK-Blue hTLR4 cell line. The results were compared with control (cells treated with PBS), while 10% DMSO is used as a positive control inducing cell lethality. Data are expressed as Mean ± SD. (****) P < 0.0001 versus the control group (PBS)

Article Snippet: HEK-BlueTM cell culture The HEK-BlueTM hTLR4 and HEK-BlueTM Null2 cells were obtained from InvivoGen, USA.

Techniques: Control, Positive Control

Journal: Advanced Biomedical Research

Article Title: Immunomodulatory Activity of Polysaccharide from Trametes gibbosa (Pers.) Fr (Basidiomycota, Fungi) Mediated by TLR4 Signaling Pathway

doi: 10.4103/abr.abr_50_22

Figure Lengend Snippet: TLR4 activation in HEK-Blue hTLR4 cells by T. gibbosa polysaccharide fraction and its synergistic effect with LPS. Data are expressed as Mean ± SD. (*) P < 0.05, (***)

Article Snippet: HEK-BlueTM cell culture The HEK-BlueTM hTLR4 and HEK-BlueTM Null2 cells were obtained from InvivoGen, USA.

Techniques: Activation Assay

Journal: Advanced Biomedical Research

Article Title: Immunomodulatory Activity of Polysaccharide from Trametes gibbosa (Pers.) Fr (Basidiomycota, Fungi) Mediated by TLR4 Signaling Pathway

doi: 10.4103/abr.abr_50_22

Figure Lengend Snippet: The concentration of IL-8 in HEK-Blue hTLR4 cells treated with T. gibbosa polysaccharide fraction and LPS. Data are expressed as Mean ± SD. (****) P < 0.0001 versus the control group (PBS)

Article Snippet: HEK-BlueTM cell culture The HEK-BlueTM hTLR4 and HEK-BlueTM Null2 cells were obtained from InvivoGen, USA.

Techniques: Concentration Assay, Control

Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: Chemical composition and pH-responsive behavior of UPS/IL-2-Fc NP (A) Schematic illustrating IL-2-Fc activity control via pH-threshold-dependent encapsulation and release. (B) Chemical structure of UPS polymers used to formulate pH-activatable IL-2-Fc nanoparticles. (C–E) pH-dependent characterization of three UPS/IL-2-Fc formulations: nanoparticle size (C), IL-2-Fc release by ELISA (D), and IL-2 activity in HEK-Blue IL-2 reporter cells (E) ( n = 3). (F) Encapsulation stability of IL-2-Fc in different UPS formulations incubated in PBS, cell culture medium, and mouse plasma at 37°C ( n = 3). (G and H) In vivo evaluation of antitumor efficacy and cytokine release in MC-38 tumor-bearing mice treated with various UPS/IL-2-Fc NPs or unencapsulated IL-2-Fc (0.75 mg/kg, n = 5). Data in (C–G) are presented as mean ± SEM. Heatmap in (H) generated from Z score normalization by column. See also .

Article Snippet: HEK-BlueTM IL-2 Cells , Invivogen , hkb-il2.

Techniques: Activity Assay, Control, Encapsulation, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture, Clinical Proteomics, In Vivo, Generated

Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: pH-responsive blocking and restoration of IL-2 receptor binding by UPS 5.3 (A) Schematic of the experimental design to assess the intrinsic interaction between UPS 5.3 micelles and proteins. (B) FPLC chromatograms showing individual proteins or UPS 5.3 /protein mixtures. (C) Molecular docking model of the DBA dimer (blue) with IL-2, highlighting predicted interaction sites. (D–F) Biolayer interferometry analysis of IL-2-Fc binding to IL-2Rα, IL-2Rβ, and IL-2Rγ, with and without UPS 5.3 NP encapsulation. (G) Schematic illustrating ex vivo assessment of IL-2-Fc binding to immune cells from various organs. (H) Quantification of IL-2-Fc binding to lymphocytes isolated from liver, lung, spleen, and tumor ( n = 5). Data are presented as mean ± SEM. See also and .

Article Snippet: HEK-BlueTM IL-2 Cells , Invivogen , hkb-il2.

Techniques: Blocking Assay, Binding Assay, Encapsulation, Ex Vivo, Isolation

Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: Tumor-specific release of IL-2-Fc by UPS 5.3 /IL-2-Fc NP (A) Schematic illustrating measurement of free and total IL-2-Fc levels and the concept of tumor-specific activation. (B) Pharmacokinetic analysis of free and total IL-2-Fc following intravenous administration of UPS 5.3 /IL-2-Fc NP or unencapsulated IL-2-Fc alone ( n = 5). (C) AUC comparison of free and total IL-2-Fc concentrations ( n = 5). (D) PET/computed tomography imaging and quantification in MC-38 tumor-bearing mice 24 h after intravenous injection of 64 Cu-labeled IL-2-Fc or UPS 5.3 /IL-2-Fc NP ( n = 3). (E) Schematic depicting the FRET-based design for assessing IL-2-Fc encapsulation status in vivo . (F) FRET-based analysis of IL-2-Fc encapsulation and release in the spleen, lung, liver, and tumor following systemic administration. Scale bar, 50 μm. Data in (B–D) are shown as mean ± SEM. See also and .

Article Snippet: HEK-BlueTM IL-2 Cells , Invivogen , hkb-il2.

Techniques: Activation Assay, Comparison, Computed Tomography, Imaging, Injection, Labeling, Encapsulation, In Vivo

Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: UPS 5.3 /IL-2-Fc NP binds cytotoxic immune cells in tumors and elicits strong antitumor responses (A) Schematic illustrating tumor-specific activation of UPS 5.3 /IL-2-Fc NP in response to severe acidity, promoting cytotoxic immune cell stimulation. (B) Treatment schedule for in vivo evaluation of NK and CD8 + T cell binding and activation. (C) Binding and activation of CD8 + T cells and NK cells by UPS 5.3 /IL-2-Fc NP (red) in MC-38 tumors, showing comparable effects to unencapsulated IL-2-Fc (blue, n = 5). (D) Dose-dependent antitumor efficacy of UPS 5.3 /IL-2-Fc NP in the MC-38 tumor model (n = 7–8). (E) Anti-tumor effect in 4T1 orthotopic tumors with starting size >400 mm 3 . UPS 5.3 /IL-2-Fc NP was administered at 2.25 mg/kg for three doses ( n = 6). (F) Combination therapy in the B16F10 model using UPS 5.3 /IL-2-Fc with either anti-PD-1 or PolySTING. UPS 5.3 /IL-2-Fc NP was administered at 2.25 mg/kg, twice (n = 5–7). Data are shown as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. See also and .

Article Snippet: HEK-BlueTM IL-2 Cells , Invivogen , hkb-il2.

Techniques: Activation Assay, Cell Stimulation, In Vivo, Binding Assay

Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: UPS 5.3 encapsulation blocks IL-2-Fc binding to NK cells in normal tissues, reducing systemic toxicity (A) Treatment regimen and assessment of cell and receptor dependency in IL-2-Fc-induced vascular leak syndrome (VLS). (B) Schematic illustrating UPS 5.3 -mediated inhibition of NK cell activation by IL-2-Fc in normal tissues. (C) Treatment schedule for in vivo evaluation of NK cell binding and activation in major organs. (D) Comparison of IL-2-Fc binding to NK cells and NK cell counts in the spleen, liver, and lung from UPS 5.3 -IL-2-Fc NP (red) and unencapsulated IL-2-Fc (blue). (n = 5–7). (E–G) Improved safety profile of UPS 5.3 /IL-2-Fc NP in C57BL/6 mice, demonstrated by stable body weight (E, n = 5), >100-fold reduction in systemic IFN-γ (F, n = 5), and absence of lung edema (G, n = 3). n = 3 for (A); n = 5 for (D–G). Data are presented as mean ± SEM. ∗∗∗∗ p < 0.0001. See also .

Article Snippet: HEK-BlueTM IL-2 Cells , Invivogen , hkb-il2.

Techniques: Encapsulation, Binding Assay, Inhibition, Activation Assay, In Vivo, Comparison

Journal: Cell Reports Medicine

Article Title: Targeting severe acidity for tumor-activatable Interleukin-2 therapy

doi: 10.1016/j.xcrm.2025.102572

Figure Lengend Snippet: UPS 5.3 NP encapsulation of IL-2-Fc leverages macrophage clearance to reduce systemic toxicity (A) Treatment regimen and histological analysis showing differential IL-2-Fc distribution in the spleen following nanoparticle or free IL-2-Fc administration. W, white pulp; R, red pulp. Scale bars, 100 μm. (B) Body weight changes in C57BL/6 mice treated with IL-2-Fc or UPS 5.3 /IL-2-Fc NP, with or without macrophage depletion using anti-CSF1R ( n = 3). IL-2-Fc dose: 0.75 mg/kg per injection. (C) PBMC-engrafted humanized mice treated with different IL-2-Fc formulations ( n = 5). (D) CD34 + cell-engrafted humanized mice treated with different IL-2-Fc formulations ( n = 5). IL-2-Fc dose for (C and D): 2.25 mg/kg per injection. (E) Circulating cytokine profiles measured in both humanized mouse models post-treatment ( n = 3). Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01. See also .

Article Snippet: HEK-BlueTM IL-2 Cells , Invivogen , hkb-il2.

Techniques: Encapsulation, Injection

Journal: bioRxiv

Article Title: Bioactive Enhanced Adjuvant Chemokine Oligonucleotide Nanoparticles (BEACONs) for Mucosal Vaccination Against Genital Herpes

doi: 10.1101/2025.07.31.667899

Figure Lengend Snippet: (A) Schematic of preparation of DNA-chemokine nanoparticles (BEACONs). (B) Hydrodynamic diameter of BEACONs (mouse) as measured by dynamic light scattering. (C) Representative negative stain TEM image showing spherical morphology of BEACONs (mouse). Scale bar, 200 nm. (D) Measurement of CXCL9 functional activity via chemotaxis assay, quantifying the number of T cells migrated across the Transwell by measuring calcein AM fluorescence. (E) TLR9 signaling assessed by measuring fold change (relative to saline) in reporter activation in HEK-mouse-TLR9 reporter cells 48 hours after treatment with free CpG-ODNs, CXCL9, or BEACONs. (F) Frequency of CD86 + BMDCs after stimulation with BEACONs compared to controls (free CpG-ODNs, CXCL9, or medium-only).

Article Snippet: Mouse TL9 reporter assay: HEK-BlueTM mTLR9 cells (InvivoGen) were seeded at 3 × 10 4 cells per well in flat-bottom 96-well plates (Corning) for 24 hours and then treated with water (vehicle control), CXCL9, CpG-ODN, or BEACONs for 48 hours.

Techniques: Staining, Functional Assay, Activity Assay, Chemotaxis Assay, Fluorescence, Saline, Activation Assay

Journal: bioRxiv

Article Title: Development of Recombinant Anti-TLR2 Antibodies and PLGA Nanoparticle-based Gene Therapy for the Treatment of Neuropathic Pain

doi: 10.1101/2025.11.13.688281

Figure Lengend Snippet: ( A ) SPR analysis showing dose-dependent binding of nb52, nb52 dimer (nb52di), scFv33, and scFv1 to immobilized recombinant TLR2. Equilibrium dissociation constants (KD) are indicated. (B) TLR2/NF-κB signaling inhibition assessed by a SEAP reporter assay. HEK-Blue™ hTLR2 cells were pretreated with nb52di or scFv33 for 1 hour and stimulated with Pam3CSK4 (0.5 ng/mL) for 48 hours. SEAP activity was quantified as OD655, and neutralization was expressed as a percentage relative to the Pam3CSK4-only controls. Data represent the mean ± SEM of 2–3 technical replicates. (C) qRT-PCR analysis of proinflammatory cytokine expression in BV2 cells. Cells were pretreated with nb52di (50 μg/mL) or scFv33 (30 μg/mL) for 1 hour and stimulated with Pam3CSK4 (50 ng/mL) for 3 hours. Expression levels of TNF-α, IL-1β, and IL-6 are shown relative to unstimulated controls. Data represent the mean ± SEM of 2 technical replicates. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test (**p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Pam3CSK4 group). (D) qRT-PCR analysis of proinflammatory cytokine expression in mouse spinal cords (L4–L6). Mice received a single intrathecal injection of Pam3CSK4 (10 ng) with and without the nb52 dimer or scFv33 (2 μg) in 10 μL of saline. Each dot represents an individual mouse. Data represent the mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (*p < 0.05, **p < 0.01 vs. Pam3CSK4+saline group).

Article Snippet: To assess the inhibitory effects of antibodies on TLR2 signaling, SEAP reporter assays were performed using HEK-BlueTM hTLR2 cells (InvivoGen, San Diego, CA, USA), which stably express human TLR2 and a SEAP reporter gene under the control of an NF-κB-inducible promoter.

Techniques: Binding Assay, Recombinant, Inhibition, Reporter Assay, Activity Assay, Neutralization, Quantitative RT-PCR, Expressing, Injection, Saline