Monensin

Nigericin (Sigma N7143) was resuspended in ethanol (20 mM) and stored at −20 °C. LPS-B5 (Invivogen tlrl-b5lps) was resuspended in ultrapure water, aliquoted, and stored at −20 °C. Monensin (Cayman Chemicals #16488) was dissolved in ethanol (10 mM) immediately before use. CL097 (Sigma SML2566) was resuspended in ultrapure water (5 mg/mL) with dropwise addition of 35% HCl, aliquoted, and stored at −80 °C. Imiquimod hydrochloride (MedChemExpress HY-B0180A) was resuspended in ultrapure water with sonication (4 mg/mL), aliquoted, and stored at −80 °C. Biotin tyramide (Iris biotech LS-3500) was resuspended in DMSO (500 mM), aliquoted, and stored at −80 °C. PMA (Sigma P1585) was resuspended in DMSO (1 mg/mL), aliquoted, and stored at −80 °C. All other chemical reagents are listed in Table S15.

Human pancreatic cancer cell line Panc-1 and MiaPaCa-2 were kindly provided by Dr. Keping Xie of the University of Texas MD Anderson Cancer Center. All cells were cultured in complete DMEM containing 10% fetal bovine serum (FBS, ThermoFisher, Waltham, MA) with 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C in 5% CO₂ as previously described^{20 (link)–23 (link)}. Monensin, gemcitabine and erlotinib were obtained from Cayman Chemical (Ann Arbor, MI) and LC Laboratories (Woburn, MA), respectively. Unless indicated, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Thermos Fisher Scientific (Waltham, MA).

BJ, U2OS, and 293T cells were from ATCC and were authenticated through short tandem repeat (STR) profiling. All relevant authentication data are publicly available from ATCC. These cell lines differ in their growth rates and morphologies. All cell lines in this study were free of contaminations from other cell lines or mycoplasma. Cells were maintained with mycoplasma reagent and potential contaminations are regularly monitored through polymerase chain reaction (PCR) detection. All cells were cultured at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8% fetal bovine serum (FBS) and penicillin/streptomycin. Cyclic [G(2’,5’)pA(3’,5’)p] or cGAMP (#CT-CGMAP, ChemieTek) was dissolved in water and stored at −20 °C. The other two STING agonists diABZI (#S8796, Selleck) and C53 (#37354, Cayman) were dissolved in DMSO and stored in aliquots at −80 °C. Monensin (#16488, Cayman) was dissolved in ethanol and stored at −20 °C. cGAMP was delivered to cells using a mild digitonin buffer (50 mM HEPES pH 7.0, 100 mM KCl, 3 mM MgCl₂, 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, 1 mM ATP, 8 μg/mL digitonin) (Woodward et al, 2010 (link)). Cells were treated with the buffer containing 1 μM cGAMP for 5-10 min and then changed back to the original culture media. All other chemicals were directly added to the cell culture media.