Fig 2: PDS triggers the activation of the cGAS–STING signaling pathway. (A) IF images of cGAS-bound micronuclei in MCF-7 cells treated with 10 µM PDS (left) and cGAS-positive micronuclei levels in untreated and PDS-treated cells (right). Data show means of three biological replicates. Significance was determined with a multiple paired t-test: **P-value <0.01. (B) Levels of cGAMP dinucleotide over time in untreated and PDS-treated MCF-7 cancer cells. cGAMP levels were determined using the Direct 2',3'-Cyclic GAMP Enzyme Immunoassay Kit. Data show means ± SEM of at least three biological replicates. The P-value (determined with the Mann–Whitney test) is 0.057 at day 4. (C) IF image of STING in MCF-7 cancer cells as indicated [H151 (2µM) is a STING inhibitor]. (D) IF images of IRF3 and pIRF3 (phosphorylated Ser396 IRF3) in MCF-7 cancer cells as indicated. ssDNA indicates cells transfected with salmon sperm DNA as a positive control. (E) Fraction of pIRF3-positive (>2 nuclear foci) MCF-7 cells at day 4. Data show means ± SEM of two biological replicates. Significance was calculated by the chi-square test: ****P-value <0.0001. Scale bar is 10 µm.

Fig 3: cGAS-STING pathway is essential for TMZ and anti-CD47 induced immune response.a Combination TMZ and anti-CD47 antibody treatment against murine GBM cells (GL261) increased the production of type I interferons in APCs. IFNA interferon a, IFNB interferon ß. n = 5, **p < 0.01, one-side ANOVA with Bonferroni post hoc correction. b Western blot showing combined TMZ and anti-CD47 antibody treatment resulted in the activation of cGAS-STING cytoplasmic DNA sensing pathways in BM APCs. c Combination TMZ and anti-CD47 antibody treatment promoted p65 expression and nuclear translocation in BM APCs that is dependent on STING. d STING activation is critical to the increased the production of NF-?B cytokine TNFa and IL1ß in APCs in the setting of combined TMZ and anti-CD47 antibody treatment. n = 6, **p < 0.01, unpaired Student’s t test. e Immunofluorescence staining of GL261 tumors implanted in WT and STING KO animals. Nuclear p-IRF3 (Ser 396) in CD45+ cells is indicative of STING signaling activation. f, g Quantification of CD45+ cell infiltration and percentage of p-IRF3 positive CD45+, C57BL/6, n = 7; knockout, n = 6. Unpaired Student’s t test. h GL261 tumor volume in C57BL/6 or STING KO mice at day 20 following control or combination TMZ + aCD47 treatment, n = 5/group. i T-cell infiltration in GL261 tumors in C57BL/6 or STING KO mice at day 20 following control or combination TMZ + aCD47 treatment measured by CD3+ cells in total DAPI count per field of view (FOV), C57BL/6, n = 9; knockout, n = 6 unpaired Student’s t test. Representative FOV for each group on the right. All error bars = mean ± standard deviation.