Extended Data Fig. 4: Induction of STING signaling by intratumoral ADU-S100.
a, KPC tumor-bearing mice were treated with/without agonist monotherapy or combination therapy, and tumors were harvested at indicated timepoints. Two tumors were pooled and lysed for western blot analysis for each treatment option at each timepoint. The activation of STING pathway was analyzed by phosphorylation of IRF3 and IFNβ expression downstream of STING. A representative western blot of two independent experiments is shown. β-actin was used as a loading control. b, KPC tumors were co-stained for IFNβ with macrophage (F4/80) and endothelial cell (CD31) markers at 4 h post monotherapy or combination therapy to evaluate STING activation in these cells. Immunofluorescence images (top panels) are shown in higher magnification in the middle and/or bottom panels. IFNβ+ macrophages and endothelial cells are indicated by green and red circles, respectively. Scale bar, 100 μm (top panels) and 20 μm (middle and bottom panels). c, ADU-S100 (10 μM) was added to the culture of KPC cells or endothelial cells (HUVEC) for indicated time, and the activation of STING signaling was analyzed by IRF3 Ser385 phosphorylation. The quantification of phosphorylated IRF3 was normalized by total IRF3. β-actin was used as a loading control. d, Leukocytes were isolated from peripheral blood or the peritoneum of normal mice and incubated in vitro with 7.2 μg/ml STING agonist ADU-S100 for 2 h at 37 °C. Phosphorylation of IRF3 in leukocyte subsets (CD11b+ myeloid cells, CD3+ cells, CD19+ cells, and NK1.1+ cells) was detected by α-phospho-IRF3 Ser385 antibody staining. Cells were permeabilized using eBioscience™ Transcription Factor Staining Buffer Set before staining. The percent fraction of phospho-IRF3+ cells in each subset of blood or peritoneal leukocytes was determined by flow cytometry. N = 2 *p < 0.05, **p < 0.01.
