Fig 2: ZIKV NS4B interacts with DHCR7 to inhibit TBK1 and IRF3 phosphorylation. A HEK293T cells were co-transfected with plasmids encoding HA-DHCR7, and plasmids encoding ZIKV non-structural proteins (FLAG-NS1, FLAG-NS3, FLAG-NS4A, FLAG-NS4B, and FLAG-NS5) or empty control plasmid for 30 ?h. The cell lysates were immunoprecipitated with anti-Flag antibody, and then analyzed by immunoblotting with indicated antibodies. B, C HEK293T cells were co-transfected with plasmids encoding HA-DHCR7, plasmids encoding FLAG-NS4B or empty control plasmid for 30 ?h. The cell lysates were immunoprecipitated with anti-HA antibody (B) or anti-FLAG antibody (C), and then analyzed by immunoblotting with indicated antibodies. D U251 ?cells were transfected with FLAG-NS4B plasmid together with plasmids encoding with HA-DHCR7. The localization of FLAG-NS4B (green), HA-DHCR7 (red), nucleus marker DAPI (blue), and merge were analyzed with confocal microscopy. Scale bar ?= ?10 ?µm. E Cell lysates from HEK293T cells transfected with HA-DHCR7 were incubated with GST protein or GST-NS4B protein which was incubated with glutathione-Sepharose beads. Mixtures were detected by Western blot with anti-HA and anti-GST antibody (top). Lysates from transfected HEK293T cells and the purified proteins were a detected by Western blot with anti-HA and anti-GST antibody (bottom). F U251 ?cells were transfected with plasmid encoding FLAG-NS4B (0, 1, 2 ?µg). The relative protein levels of DHCR7, FLAG-NS4B and GAPDH were detected by Western blot. G, H HEK293T cells were co-transfected with NS4B-expressing plasmid or empty vector, together with TBK1-coding plasmid (F), IRF3-expressing plasmid and plasmids encoding RIG-I (G). Cells were harvested at 24 ?h post-transfection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-FLAG, and anti-GAPDH]. I DHCR7-silencing U251 (sh-DHCR7) and sh-NTC U251 ?cells were transfected with NS4B-expressing plasmid or empty vector. Cells were harvested at 6 ?h post-transfection and subjected to Western blot with indicated antibodies [anti-TBK1, anti-P-TBK1(S172), anti-IRF3, anti-P-IRF3(S396), anti-FLAG, and anti-GAPDH]. Data are representative of three independent experiments.

Fig 3: The small molecule ACY738 increases ARIH1 protein levels and enhances PD-L1 blockade therapy in 4T1 tumor models.a High-throughput screening of 8207 drug or drug candidates was performed to screen for ARIH1 enhancers that increase ARIH1 levels. After treatment with the drugs at 5 μM for 24 h, luciferase as a reporter to detect ARIH1 protein levels following exposing to the substrate (10 μM) in 384-well plates. Hit compounds are shown as red dots. b Immunoblots (IB) analysis for the ARIH1 levels in MDA-MB-231 and 4T1 cells treated with 1 µM ACY738 at the indicated time points. The numbers under the blots represent the gray scale quantification (ARIH1/Actin and ARIH1/Tubulin). c Tumor growth curves from subcutaneous injection of 5 × 105 4T1 cells in the female BALB/c mice (6–8 week old) treated with vehicle, anti-PD-L1 alone, ACY738 alone, and ACY738+anti-PD-L1. n = 6 mice/group. Data represent means ± SEM, **P < 0.01 (P = 0.0012), ***P < 0.001 (P = 0.0005), ****P < 0.0001. d–f. Representative image, final tumor weights, and survival curves of tumor bearing mice with the indicated treatments. n = 6 mice/group. Data represent means ± SEM, e *P < 0.05 (P = 0.0151), ***P < 0.001 (P = 0.0010). Log-rank test (f), *P < 0.05 (P = 0.0200), **P < 0.01. g, h Representative images and quantification of spontaneous lung metastases and lung weights of BALB/c mice (6–8 week old) at day 16 with the indicated treatments after intravenous injection of 1 × 105 4T1 cells. Scale bar, 400 μm. n = 5 mice/group. Data represent means ± SEM, **P < 0.01, ***P < 0.001 (P = 0.0008), ****P < 0.0001. i Quantification of tumor-infiltrating CD8+ T cells of the mice as in c. n = 6 mice/group. Data represent means ± SEM, **P < 0.01 (P = 0.0018), ***P < 0.001. j, k The CD8 and GzmB IHC staining (j) was performed in tumors of the mice as in c. The percent of each expression pattern was quantified (k). Scale bar, 60 μm. n = 6 mice/group. Data represent means ± SEM, ***P < 0.001, ****P < 0.0001. l IB analysis of total and phospho STING (S365), total and phospho TBK1(S172), and ARIH1 in tumor lysates of the mice as in c. For c data, Two-way ANOVA test. For e, h-i, and k data, One-way ANOVA test. Data shown in a, b and i are representative of three independent experiments. Source data are provided as a Source Data file.

Fig 4: PA activated STING-TBK1-IRF3/NF-?B pathway and induced inflammation in MH-S and RAW264.7 macrophages.MH-S cells were treated with PA (0.6 mM) for 24 h. a–d Representative western blots of the STING (a), p-TBK1 (b), TBK1 (b), p-IRF3 (c), IRF3 (c), p-p65 (d) and p65 (d) in PA-treated MH-S cells. e–h Group data of STING (e), p-TBK1/TBK1 (f), p-IRF3/IRF3 (g) and p-p65/p65 (h) fold change in equal amounts of protein extracts of control (n = 4 biological repeats) and PA-treated (n = 5 biological repeats) MH-S cells. RAW264.7 macrophages were treated with PA (0.6 mM) for 24 h. i–l Representative western blots showed the STING (i), p-TBK1 (j), TBK1 (j), p-IRF3 (k), IRF3 (k), p-p65 (l) and p65 (l) in PA-treated RAW264.7 macrophages. m–p Group data of STING (m), p-TBK1/TBK1 (n), p-IRF3/IRF3 (o) and p-p65/p65 (p) fold change in equal amounts of protein extracts of control (n = 4 biological repeats) and PA-treated (n = 5 biological repeats) RAW264.7 macrophages. q–r ELISA analysis showed that the protein levels of TNF-a, IL-6 and IFNß in MH-S cells (q) and RAW264.7 macrophages (r) were significantly increased after PA treatment. n = 3 biologically independent experiments. Data represent mean ± SD. Statistical significance was determined using two-tailed Student’s t test.

Fig 5: ARIH1 promotes the STING pathway activation.a Heatmap for the qRT-PCR expression data of genes for 10 cytokines and 11 chemokines in CTRL and Arih1-WT-OE 4T1 tumors (n = 3 per group). b qRT-PCR analysis for the gene expression of Ifnb1, Ifna2, Il-6, and Ccl5 as in a. n = 3 per group. Data represent means ± SEM, **P < 0.01 (P = 0.0013), ***P < 0.001, ****P < 0.0001. c Immunofluorescence analysis of dsDNAs and cGAS in CTRL and Arih1-WT-OE 4T1 cells. The nuclei were stained with DAPI. Representative confocal images and quantitative data are shown. CTRL group (n = 31 cells), Arih1-WT-OE group (n = 43 cells). Scale bar, 10 µm; insets: Scale bar, 2 µm. Data represent means ± SEM, **P < 0.01 (P = 0.0042), ****P < 0.0001. d Immunoblots of markers in the STING pathway including total and phospho STING (S366 sites for human; S365 sites for mouse), total and phospho TBK1(S172) in lysates collected from breast cell lines (MCF-7 and MDA-MB-231) and 4T1 tumors (from Fig. 2a) with indicated treatments. e Immunofluorescent staining of IRF3 in CTRL and Arih1-WT-OE 4T1 cells and their quantifications. The nuclei were stained with DAPI. Scale bar, 10 µm; insets: Scale bar, 5 µm. Data represent means ± SEM, ****P < 0.0001. Each dot in the graph represents the percentage of counted nucleus IRF3 cells in each sample, and the total number of counted cells in each group is as follows: CTRL group (n = 135 cells), Arih1-WT-OE group (n = 147 cells). For b, c, e data, Two-tailed t-test. Data shown in c–e are representative of three independent experiments. Source data are provided as a Source Data file.