Forskolin

The adenylate cyclase activator forskolin [HY-15371], PDE4 inhibitor zardaverine [HY-15485], adenylate cyclase inhibitor SQ22536 [HY-100396] and BET inhibitor JQ1 [HY-78695] were purchased from MedChemExpress. Human IFN-gamma [285-IF) were purchased from R&D systems (Minnesota, USA); forskolin, zardaverine, and JQ1 were dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C.

PPT, DPN, MPP, PHTPP, E₂, forskolin, genistein, rottlerin, wortmannin, GSK-7975A, ML-9, SKF-96365, CPA, and BAPTA-AM were purchased from MedChemExpress and dissolved in DMSO. CCh and indomethacin were purchased from Sigma-Aldrich. CCh was dissolved in ultrapure water. indomethacin was dissolved in anhydrous alcohol. All salts were supplied by Sangon Biotech and dissolved in ultrapure water.

Compounds and reagents include: lactate (#L6402, Sigma-Aldrich, USA), phorbol 12-myristate 13-acetate (PMA, #S7791, Selleck Chemicals, USA), LDHA inhibitor, sodium oxamate (SO, #6871, Selleck Chemicals, USA), MCT inhibitor, a-cyano-4-hydroxycinnamate (CHC, #S8612, Selleck Chemicals, USA), HMGB1 inhibitor, glycyrrhizin (#HY-N0184, MedChemExpress, USA), adenylate cyclase inhibitor, SQ22536 (#HY-100396, MedChemExpress, USA), PKA inhibitor, H89 (#HY-15979, MedChemExpress, USA), Forskolin (#HY-15371, MedChemExpress, USA), rHMGB1 (#ab167718, Abcam, USA).

Hormones and inhibitors used in culture for in vitro experiments were as follows: Glucagon (Sigma-Aldrich, catalog #G2044) was prepared in 0.05 M acetic acid (Sigma-Aldrich, catalog # 6283) at a concentration of 1 mg/ml. Forskolin (MedChem Express, catalog # HY-15371) was prepared in DMSO (Sigma-Aldrich, catalog # 2650) at a stock concentration of 10 mM. 666–15 (Sigma-Aldrich, catalog # 5383410001) was prepared in DMSO at a stock concentration of 10 mM. Palbociclib (Selleck Chemicals, catalog # S1116) was prepared in DMSO at a stock concentration of 10 mM. GSK126 (Selleck Chemicals, catalog # S7061) was prepared in DMSO at a stock concentration of 10 mM. LBH589 (Selleck Chemicals, catalog # S1030) was prepared in DMSO at an initial concentration of 10 mM and diluted to 0.1 mM for a working stock solution. Decitabine (Selleck Chemicals, catalog # S1200) was prepared in DMSO at a stock concentration of 10 mM. Sorafenib (Selleck Chemicals, catalog # S1040) was prepared in DMSO at a stock concentration of 10 mM. UNC0642 (Selleck Chemicals, catalog # S7230) was prepared in ethanol (Decon labs) at a stock concentration of 10 mM.

Uric acid sodium was purchased from Sigma Aldrich (St. Louis, USA). Forskolin and SQ22536 were purchased from MedChemExpress (USA). Phorbol 12-myristate 13-acetate (PMA), propidium iodide (PI), 4’, 6-diamidino-2-phenylindole (DAPI), and Antifade Mounting Medium were provided by Keygen biotech (Nanjing, China). siRNA for transfection was obtained from Genepharma (Shanghai, China). Lipofectamine 2000 was derived from Invitrogen. Caspase-1 Detection Kit was supplied from ImmunoChemistry Technologies (USA). NLRP3 (bs-10021R), ASC (bs-6741R), caspase-1 (bs-10442R), and β-actin (bs-0061R) antibodies for western blot were obtained from Bioss (Beijing, China). NLRP3 (sc-34410) and ASC (sc-22514-R) primary antibodies used for immunofluorescence were obtained from Santa Cruz Biotechnology (USA). Donkey Anti-Goat IgG H&L (Alexa Fluor 488) (ab150129) and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 647) (ab150075) second antibodies used for Immunofluorescence were obtained from Abcam (USA). All cell culture supplement components were obtained from Gibco (Waltham, MA, USA).

The human MPNST cell line sNF96.2 and pNF cell line ipNF05.5 were purchased from the American Type Culture Collection (ATCC, USA). All cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and maintained at 37°C in 5% CO₂. Forskolin, 3-isobutyl-1-methylxanthine (IBMX) and H89 were purchased from MedChem Express (MCE, USA). Cells were treated with 25 µM Forskolin and 100 µM IBMX for 20 min; this treatment is designated F/I in the text and figures. The protein kinase A (PKA) inhibitor H89 was applied at 10 µM 20 min prior to treatment with F/I.