Fig 2: PP VII induces CD8+ T cell infiltration and inactivates STAT3 transcription intratumorally dependent on STING. (a,b) IHC analysis (b) for indicated tumors is depicted as mean + SD of ten random fields in 3 samples from pools of 3 mice showing one representative image (a). Scale bars denote 20 or 50 µm. ***p < 0.001, **p < 0.01, *p < 0.05, ns no significant.

Fig 3: Treatment with Mn alone induces phosphorylation of TBK1 via an ATM-TBK1 phosphorylation signaling pathway(A) Macrophages were treated with MnCl2 at 25 μM. The cell lysate was collected at the indicated time points after MnCl2 treatment, and Western blotting was performed using anti-p-STING, anti-STING, anti-p-TBK1, anti-TBK1, and anti-β-actin antibodies. The untreated sample was collected at 24 h after cell seeding. One representative experiment of at least three independent experiments is shown.(B) HEK cells or STING-knockout HEK (STING-KO) cells were treated with MnCl2 at 25 μM, and the cell lysate was collected at the indicated time points. Western blotting was performed using anti-p-TBK1 and anti-β-actin antibodies. The untreated sample was collected at 24 h after cell seeding. One representative experiment of at least three independent experiments is shown.(C) Macrophages were treated with different concentration of ATM inhibitor for 30 min before Mn was added to the medium, and the cells were stimulated by DNA transfection at 24 h after Mn pretreatment. Total RNA was extracted at 24 h after DNA transfection. Relative IFNL1 mRNA expression level was measured using real-time RT-PCR assay. One representative experiment of three independent experiments is shown. And each was done as triplicate. Data are shown as mean ± SD (n = 3).(D) Macrophages was treated first with different concentration of ATM inhibitor for 30 min. And then Mn2+ at the concentration of 25 μM was added to the cells. The whole cell lysate was collected after 24 h Mn pretreatment. WB was performed to detect P-TBK1, TBK1, P-ATM and ATM proteins. And anti-GAPDH antibody was also included as the loading control for all the samples. One representative experiment of at least three independent experiments is shown.(E) Macrophages were transfected with siRNA targeting ATM (siATM) or control siRNA (siCtrl) at 25 nM, and then the cells were treated by Mn2+ at 25μMat 24 h after siRNA transfection. The whole cell lysate was collected at 24 h after Mn treatment. WB was performed to probe anti-ATM and anti-p-TBK1, anti-TBK1 antibodies, anti-β-actin is also included as a loading control for all the samples (the 1st panel). One representative experiment of at least three independent experiments is shown. The WB image was further analyzed using Image J to quantify the knocking-down efficiency of ATM expression (the 2nd panel), and the inhibition of phosphorylated TBK1 under the condition of ATM was knocked down (the 3rd panel). Data are shown as mean ± SD of three independent analyses (n = 3). ∗∗∗∗p< 0.0001 (One-way ANOVA) was indicated in the figures.(F) Macrophages were treated with siATM for 24 h before Mn was added to the medium, and the cells were stimulated by DNA transfection at 24 h after Mn treatment. Total RNA was extracted at 24 h after DNA transfection. Relative IFNL1 mRNA expression level was measured using real-time RT-PCR assay. One representative experiment of at least three independent experiments is shown. Each was done as triplicate. Data are shown as mean ± SD (n = 3). ∗∗∗∗p< 0.0001 (One-way ANOVA) was indicated in the figure.

Fig 4: DDX56 promotes endogenous cGAS expression. (A) Empty vector (EV, 1 μg) or pCMV-Myc-DDX56 (1 μg) plasmid was transfected into PK15 cells for 24 h, and cells were collected for western blot analysis using specific antibodies to detect the expression of cGAS(58 kDa), STING(42 kDa), TBK1(84 kDa) and IRF3(55 kDa). GAPDH (37 kDa) was used as a loading control. (B) PK15 cells were transfected with empty vector (EV, 1 μg) or pCMV-Myc-DDX56 (1 μg) plasmid for 24 h, then cells were collected for cellular RNA extraction. RT-qPCR was performed for IFN-β mRNA detection. Data were presented as mean ± SD of three independent experiments, measured in technical duplicates. NS stands for no significance. (C) PK15 cells were transfected with siRNA targeting MAVS (siRNA Mixture, siMAVS) for 24 h, then an empty vector (EV, 1 μg) or pCMV-Myc-DDX56 (1 μg) plasmid was transfected into above cells for another 24 h. Before being collected, cells were stimulated with 1 μg ISD for 12 h. Cellular proteins were extracted and subjected to western blotting for MAVS (56 kDa), cGAS (58 kDa), and Myc tagged DDX56 (65 kDa) detection. β-actin (42 kDa) served as loading control.

Fig 5: Increased chromosome segregation errors upon PRC1 inhibition.a Volcano plots showing the top differentially expressed pathways in low-risk UM cells (92.1) treated with PRT vs DMSO for 24 h; evaluated across biological triplicates. Normalized enrichment score (NES) for selected genesets shown as a function of significance, -log10(FDRq); FDRq, Bonferroni corrected p value. An unfiltered list of all significant gene signatures is provided in Supplementary Data File 5. b Examples of UM cells in anaphase stained for DAPI (blue) and centromeres (red); demonstrating different patterns of chromosome segregation errors. Additional patterns of missegregation are shown in Supplementary Fig. 9b. Representative images from biological triplicates. c Abundance of chromosome segregation error patterns during anaphase in low-risk UM cells (92.1) upon PRT-treatment as a function of time. Statistical significance tested using two-sided unpaired Student’s t test. Source data are provided as a Source Data file. d Abundance of chromosome segregation error patterns during anaphase across UM cell lines, arranged from left to right based on their GEP2 score. Source data are provided as a Source Data file. e Western blot of H2AK119Ub, STING, RING1, and RNF2 relative to Actin in Mel285 cells upon RING1 and RNF2 knockout. Data representative of biological triplicates. f Abundance of chromosome segregation error patterns during anaphase in UM cells (Mel285.Cas9) upon RNF2 knockout. Statistical significance tested using two-sided unpaired Student’s t test. Source data are provided as a Source Data file. Stacked bars, mean of each missegregation pattern; error bars, standard error of the mean across experimental triplicates (c–f).