Fig 2: Impairment of TBK-1 activity compromises unfolded protein response (UPR) signaling. (A) Immunoblotting depicting expression of type I IFN response drivers in BV-2 cells treated with 50 nM of bortezomib. Cells were pretreated for 1 h with 1 µM of the TBK1 inhibitor—BX795—or with dimethyl sulfoxide (DMSO) as a control. Quantitative reverse transcription PCR (RT-PCR) analysis of (B) Ifnß1 and (C) Cxcl-10 messenger RNA (mRNA) levels in BV-2 cells. Mean ± SEM (n = 3); *P < 0.05, unpaired two-sided t-test. (D) Expression of C/EBP homologous protein 10 (CHOP) and activating transcription factor 6 (ATF6) transcription factors in BV-2 after the TANK-binding kinase 1 (TBK1) inhibition and treatment with 50 nM bortezomib. (E) Quantitative RT-PCR analysis of Il-6 mRNA level in BV-2 cells after TBK1 inhibition. Mean ± SEM (n = 3).

Fig 3: BSA-Man@Mn2+-Ft@Lap nanoassembly was selectively activated in solid tumors to induce potent immune response for tumor suppression.a, b, d, e Panels a, d are the systemic distribution of the samples at 6/12/18 h post intravenous injection. I: Non-dissociable amide-ligated BSA-Man@Mn2+-Ft@Lap@Cy5; II: Tumor-responsive Schiff base-ligated BSA-Man@Mn2+-Ft@Lap@Cy5. Scale bar = 2 cm; panels b, e show the fluorescence distribution in major organs and tumors after the euthanasia. Scale bar=1 cm; panels c, f are the corresponding quantitative analysis results in each group. g In vivo bioluminescence analysis of 4T1-Luc tumor-bearing mice after different treatments. (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn2+-Ft, and (V) BSA-Man@Mn2+-Ft@Lap. Scale bar = 3 cm. h Tumor size changes during the incubation period after different treatment. i Survival analysis of mice after treatment. j Western blot analysis on the expression of damage-associated molecular patterns (HMGB1 and CRT), apoptosis markers (BAX, Bcl-2), and key cGAS/STING signaling pathway makers (STING, p-STING, IRF-3, p-IRF-3, TBK1, and p-TBK1) in tumors from 4T1 tumor-bearing mice after treatment with different samples. k H&E staining and TUNEL staining results of tumor tissue samples after different treatment. (I) control, (II) BSA-Man-Ft, (III) BSA-Man-Ft@Lap, (IV) BSA-Man@Mn2+-Ft, and (V) BSA-Man@Mn2+-Ft@Lap. In vivo western blot and histological experiments in panels j and k were repeated three times independently with similar results. Data are presented as mean values ± SEM (n = 3 mice for panels c and f, n = 5 mice for panel h). Statistical analysis for panels c and f was carried out via one-way ANOVA method. * Indicates significance at p < 0.01, *** indicates significance at p < 0.001, ****indicates significance at p < 0.0001. Source data are provided as a Source data file.

Fig 4: Western blotting analysis of phospho-IRF3 (b), TBK1 (c), and NF-κB P65 (d), ICAM1 (e), STING (f) and INF-β (g) in RMECs after stimulation with 1 μg of mtDNA for different times. Western blotting analysis of IRF3 and NF-κB P65 levels in the cytoplasm and nuclei of RMECs after stimulation with 1 μg of mtDNA for different times. h, j, m Amounts of NF-κB P65 and IRF3 in the cytoplasm. k, i, l Amounts of NF-κB P65 and IRF3 in the nuclei. ∗∗P < 0.01, n = 3 biological repeats in each group

Fig 5: STING adaptor and TBK1 kinase are required for the antiviral response of cytosolic DNA sensing pathway.MSCs were transfected with poly(dA:dT) (0.5 μg/ml) for 1 hr, and the subcellular distribution of STING and phosphorylated TBK1 were analyzed by immunofluorescence microscopy (a). Phosphorylation of TBK1 kinase was detected by Western blot (b). MSCs were transfected with siSTING for 48 hr (c)-(e) or pretreated with BX795 for 1 hr (f)-(h), and then transfected with poly(dA:dT) (0.5 μg/ml), followed by MHV-68 infection (MOI 0.1). Protein levels of STING and phosphorylated IRF3 were analyzed by Western blot (c), (f). Data are representative of three experiments with similar results. The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (d), (g). The virus titers in the supernatant were determined by plaque assay at 24 hr post-infection (e), (h). Real-time PCR data are shown as mean ± SEM of three independent experiments. **, p < 0.01; ***, p < 0.001.