Adu s100

The CT-26 adenocarcinoma cells (3 × 10⁵) were injected subcutaneously (SC) into the right flank of the mouse in 100 µL of phosphate-buffered saline (PBS). Every other day, the tumor size was measured with a digital caliper for a total of 30 days, and the tumor volume was calculated using the equation 1:
V = (L × W²)/2 (1)
where the letter “L” represents the large diameter and “W” represents the small diameter of the tumor (49 (link), 50 (link)). The mice were divided into five groups (n = 7), as depicted in Table 1, and treatment with ADU-S100 (MedChemExpress, South Brunswick Township, NJ, USA) and CpG ODN1826 (InvivoGen, San Diego, CA, USA) was administered intratumorally on days 10 and 16 post tumor inoculation. The mice were euthanized on day 30 post-tumor induction and their body weights were recorded. Subsequently, the tumors were extracted and weighed. After conducting a necropsy of the mice, samples of their tumor, spleen, and liver tissues were taken. The tumor inhibition rate was calculated using the equation 2 (6 (link)):
Tumor inhibition rate (%) = [(mean tumor weight of control group − mean tumor weight in treated group)/mean tumor weight of control group] × 100% (2).
The spleen index was calculated using the equation 3 (51 (link)):
Spleen index = weight of spleen (mg)/body weight (g). (3)

The indicated cells were stimulated with either ISD (Sangon Biotech), HSV-1 (Provided by Dr. Zhengfan Jiang), HSV60mer (Sangon Biotech), SATE-cddA^{70 (link)}, 2’3’-cGAMP (Invivogen, Cat# tlrl-nacga23-1), ADU-S100 (MedChemExpress, Cat# HY-12885A) or Poly(I:C) (Invivogen, Cat# tlrl-pic) after XQ2 or XQ2B treatment. Total RNA was isolated from indicated cells that incubated with the drugs for indicated time or mice tissues by using NucleoZOL reagent (MACHEREY-NAGEL) according to the manufacturer’s instructions. The quantifications of mRNA levels were carried out by real-time PCR using a PerfectStart® Uni RT&qPCR Kit (TransGen Biotech) on the QuantStudio 5 qPCR machine (Applied Biosystems). Samples were carried out in triplicate and the target C_T values were normalized to GAPDH C_T values. Primers of qPCR used in this study can be found in Supplementary Table S2.

Morphine was purchased from Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co. LTD (Shenyang, Liaoning, China). Fentanyl and sufentanil were purchased from RenFu Pharmaceutical Co. (Yichang, Hubei, China). The compound 48/80, chloroquine (CQ) and BX795 were from Sigma-Aldrich (St. Louis, MO, USA). Diphenylcyclopropenone (DCP) was from Shanghai Aladdin Biochem Technology Co., Ltd (Shanghai, China). DMXAA and ADU-S100 were from MedChemExpress (Shanghai, China). Recombinant IFN-α, recombinant IFN-β, IFN-α neutralizing antibody (anti-IFN-α) and IFN-β neutralizing antibody (anti-IFN-β) were from PBL Assay Science (Piscataway, NJ, USA). For intrathecal (i.t.) injection, spinal cord puncture was made with a 30 G needle between the L₄ and L₅ level to deliver drugs (5 µl) to the cerebral spinal fluid [21 (link)].

NVB (Selleckchem, NSC 2382) was reconstituted in DMSO at the indicated concentrations for in vitro studies and in PBS at 75 mg/kg for in vivo studies. ART558 (MedChemExpress, HY-141520) was reconstituted in DMSO and used at a concentration of 1 μM in vitro. ADU-S100 (MedChemExpress, MIW815) was reconstituted in DMSO and used at a concentration of 10 μM in vitro.