hek dualtm htlr3 reporter cells  (InvivoGen)

Journal: bioRxiv

Article Title: Developing potent and selective TBK1 molecular glue degraders for cancer immunotherapy

doi: 10.64898/2026.01.30.702304

Figure Lengend Snippet: (A) Schematic representation depicting the role of TBK1 in regulation of TNFR1- and IFN-induced signalling and cell death. (B) IRF pathway activation was assessed using a secreted Lucia luciferase reporter assay in HEK-Dual TM hTLR3 cells. Cells were preincubated with 1 µM CCT412020 (12 h) prior to stimulation with poly (I:C) (1 µg/ml) for 6 h. IRF-dependent signalling was quantified by measuring secreted Lucia luciferase activity in the culture supernatant using a luminometer. (C) NF-κB pathway activity was measured using a SEAP reporter assay in HEK-Dual™ hTLR3 cells. Cells were pre-incubated with the 1 µM CCT412020 for 12 h prior to stimulation with poly(I:C) (1 µg/mL) for 6 h. NF-κB-dependent SEAP activity was quantified by measuring absorbance at 595 nm. (D) TNF induced NF-κB pathway activity was measured using a SEAP reporter assay in HEK-Dual™ hTLR3 cells. Cells were pre-incubated with the 1 µM CCT412020 for 12 h prior to stimulation with TNF (10 ng/mL) for 6 h. NF-κB–dependent SEAP activity was quantified by measuring absorbance at 595 nm. (E) Cell viability was assessed using a CellTiter-Glo (CTG) assay in the breast cancer cell lines MCF7. Cells were pre-treated with CCT412020 (1 µM) and IFNβ (1 ng/ml) for 24 h, after which TNF (10 ng/ml) was added either as a single agents or in combination for additional 44 h. Viability was quantified following treatment to evaluate the impact of TBK1 degradation alone or in combination with pro-inflammatory cytokine signalling. (F) Dose-response analysis of CCT412020 in MCF7 cells in the presence or absence of IFNβ (1 ng/ml) for 48h. Cell viability was assessed using a CellTiter-Glo assay, and DC₅₀ values were determined from a representative experiment. (G) Cell viability in HCC38 cells was assessed using a CellTiter-Glo (CTG) as in (E) . (H) Cell viability in BT549 cells was assessed using a CellTiter-Glo (CTG) as in (E) . (I) Cell viability was assessed using a CellTiter-Glo (CTG) assay in MC38 and MC38 hCRBN cells. Cells were treated with CCT412020 for 18 h in the presence or absence of TNF. Viability was quantified following treatment to evaluate the impact of TBK1 degradation and the contribution of human CRBN expression to TNF-mediated cytotoxic responses. (J) Long-term clonogenic survival (7 days) was assessed in MC38 and MC38 hCRBN cells following treatment with CCT412020 in the presence or absence of TNFα. Cells were treated as indicated and allowed to grow for colony formation. Representative images of stained colonies in culture wells are shown, and clonogenic survival was quantified (K) from scanned plates and plotted as indicated.

Article Snippet: HEK-DualTM hTLR3 reporter cells (InvivoGen; Cat. #hkd-htlr3) were seeded in 96-well plates at a density of 1.0 × 104 cells per well and allowed to adhere overnight.

Techniques: Activation Assay, Luciferase, Reporter Assay, Activity Assay, Incubation, CTG Assay, Glo Assay, Expressing, Staining

Journal: Nature Communications

Article Title: Discovery of a small molecule TLR3 agonist adjuvant

doi: 10.1038/s41467-025-66878-3

Figure Lengend Snippet: A , B Wild-type and MyD88-, TBK1-, and IRF3-knockout THP-1 cells were stimulated with 25 µM PVP-057 for 24 h. Results are shown as mean percentage activity of 100 ng/mL TNF for NF-κB signaling ( A ) and of 1 µg/mL IFNβ for IRF-Lucia signaling ( B ), with the mean activity in the parent cell line indicated by the dotted line. Error bars represent SEM. Welch ANOVA with post-hoc Dunnett’s test against the parent cell line were applied. N = 7 technical replicates per cell condition ( A , B ). HEK-hTLR3-overexpressing cells and their parental HEK-Null1 cells along with HEK-hTLR4-overexpressing cells ( C ), and wild-type and TRIF knockout THP-1 cells ( D , E ) were stimulated with PVP-057 for 24 h in an 8-point, 1:2 dilution scheme with a top concentration of 50 µM. N = 4, 7, and 8 technical replicates for Null1, hTLR3, and hTLR4, respectively, at each concentration ( C ). N = 8 technical replicates at all concentrations except 50 µM ( N = 5) ( D , E ). Results are shown as mean fold activity over DMSO; error bars represent SEM. Statistical comparisons employed the Mann–Whitney U test. NS p > 0.05 , * p ≤ 0.05, * *p ≤ 0.01, ** * p ≤ 0.001, and **** p ≤ 0.0001. Source data are provided as a Source Data file.

Article Snippet: F HEK-Blue hTLR3 cells and their parental HEK-Blue Null1 cells (InvivoGen) were stimulated with a dose-titration of each batch of PVP-057, and NF-kB was quantified 22 h post-stimulation.

Techniques: Knock-Out, Activity Assay, Concentration Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Discovery of a small molecule TLR3 agonist adjuvant

doi: 10.1038/s41467-025-66878-3

Figure Lengend Snippet: A (i) 3,5-dimethoxybenzoyl chloride, pyridine, 0–120 ᵒC, 10 min, pH 2; (ii) 10% NaOH, EtOH, RT, 1 h, pH 2; (iii) 2-amino-6-ethoxybenzothiazole, EDC, HOAt, 10:1 MeCN/DCM, RT 24 h. The structure and purity of the product were determined by UPLC-MS and 1 H-NMR (detailed synthesis and analytical information in supplementary). B PVP-057 was synthesized in increasing quantities and the final product tested for purity level variations as measured by UPLC-MS at four increasing amounts of scaled-up production. C Table summarizing the physicochemical properties of known and well-characterized TLR3 agonists. For some or all nucleotide agonists, the molecular weight is given as a referenced estimate due to the constitutional heterogeneity of these biologics. D – F Four batches of PVP-057 were synthesized separately, by four different chemists. THP-1 Dual cells (InvivoGen) were stimulated with 25 µM of each batch of PVP-057, and induction of the IRF ( D ) and NF-kB ( E ) pathways was quantified 22 h post-stimulation. N = 2 technical replicates, averaged per batch. Data are presented as mean ± SEM. F HEK-Blue hTLR3 cells and their parental HEK-Blue Null1 cells (InvivoGen) were stimulated with a dose-titration of each batch of PVP-057, and NF-kB was quantified 22 h post-stimulation. N = 2 technical replicates. Source data are provided as a Source Data file.

Article Snippet: F HEK-Blue hTLR3 cells and their parental HEK-Blue Null1 cells (InvivoGen) were stimulated with a dose-titration of each batch of PVP-057, and NF-kB was quantified 22 h post-stimulation.

Techniques: Synthesized, Molecular Weight, Titration