Fig 1: Interplay between TRIM3 and IFN-β establishes a reciprocal feedback loop in NSCLC.A TRIM3 mRNA expression was analyzed by qPCR in H1299, PC-9, H2030, H2228, H1944, 95-D and A549 cells following stimulation with 100 ng/mL IFN-β (n = 4). Significance was assessed using Student’s t test. B–D TRIM3 protein and mRNA levels were measured in A549 and H1299 cells treated with IFN-β ± 20 ng/mL BMS-986165 or IFNAR1 and IFNAR2 knockout. Statistical significance was analyzed using one-way ANOVA. E–H IFNB1 mRNA levels were quantified by qPCR in A549 and H1299 cells with TRIM3 modulation (n = 4). Significance was assessed using Student’s t test. I–L Luciferase reporter assays were performed in A549 and H1299 cells transfected with IFNB1 promoter luciferase constructs (n = 4). Significance was assessed using Student’s t test. *p < 0.05; **p < 0.01.
Fig 2: TRIM3 ubiquitinates TLR3 to induce IFN-β secretion.A Co-IP coupled with mass spectrometry (IP‒MS) was performed in TRIM3-overexpressing H1299 cells. Immunoprecipitated proteins were quantified by unique peptide counts to identify the top 100 candidates against IFNB1-associated proteins. B, C Co-IP analysis (IP-Flag) validated the physical interaction between TRIM3 and TLR3 in A549 and H1299 cells. D, E Co-IP analysis (IP-Myc) validated the physical interaction between TRIM3 and TLR3 in A549 and H1299 cells. F The interaction between TRIM3 and TLR3 (TRIM3/TLR3; red) was analyzed by PLA in A549 and H1299 cells. G Domain mapping of TRIM3 was pictured. Co-IP between TRIM3 deletion mutants (ΔRING, ΔB-box, ΔCC and ΔNHL) and TLR3 was performed in H1299 cells. H Ubiquitination assays using HA-ubiquitin and TRIM3 catalytic mutants (C22/25S) were performed in A549 and H1299 cells. I WB was performed to detect TBK1 (Ser172) and IRF3 (Ser396) phosphorylation in WT TRIM3 vs. mutant-expressing cells under 20 ng/mL poly(I:C) stimulation. J ELISA was performed to quantify TRIM3-dependent IFN-β induction in A549 and H1299 cells (n = 4). Statistical significance was analyzed using one-way ANOVA for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001.
Supplier Page from TargetMol for IFN-beta Protein, Human, Recombinant