hekbluehtlr3  (InvivoGen)

Journal: Nucleic Acids Research

Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

doi: 10.1093/nar/gkag328

Figure Lengend Snippet: PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by Lipofectamine 3000 (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .

Article Snippet: RAW-Dual, HEK-Blue mTLR9, HEK-Blue hTLR3, HEK-Blue hTLR7, and HEK-Lucia RIG-I were purchased from InvivoGen.

Techniques: Activation Assay, In Vitro, Transfection, Expressing, High Molecular Weight, Standard Deviation, Analysis

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