3 isobutyl 1 methyl xanthine (ibmx)

IBMX was obtained from Enzo Life Sciences. NorBNI, U50488, and SNAP were obtained from Tocris Biosciences. DNQX, picrotoxin, strychnine, and 6β-naltrexol were obtained from Sigma-Aldrich. SP600125 was obtained from Calbiochem.

INS-1E cells were washed with Krebs-Ringer Buffer (135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.4 mM K₂HPO₄, 5.5 mM glucose (VWR, 10117), 20 mM HEPES (Thermo Fisher Scientific, 15630–056), pH4.7) and pre-incubated in buffer for 30 min. Following further washing, cells were incubated at 16.7 mM glucose in the presence of 500 µM IBMX (Enzo Life Sciences, BML-PD140-0200) with or without 100 nM Ex4 for 15 min before extraction into 200 µl of 0.1 mM HCl-0.5% Triton X-100 (Sigma Aldrich, X100). cAMP was determined using the Direct cAMP ELISA kit (Enzo Life Sciences, ADI-900-066) accordingly to the manufacturer’s instructions.

MG132 (Selleckchem, S2619), gefitinib (Selleckchem, S1025), nigericin (InvivoGen, tlrl-nig/NIG-36-01), brefeldin A (Cell Signaling Technology, 9972S), FTY720 (Santa Cruz Biotechnology, sc-202161A), IBMX, concanamycin A (Enzo Life Sciences, ALX-380-034-C025), tetrandrine (Selleckchem, S2403), U18666A (Cayman Chemical, 10009085), ETP-46464 (Selleckchem, S8050), JIB-04 (Tocris, 4972), nitazoxanide (COVID Box, MMV688991), ketoconazole (COVID Box, MMV637533), AG-1478 (Selleckchem, S2728), caffeic acid (Selleckchem, S7414), thapsigargin (Cell Signaling Technology, 1278S), staurosporine (Cell Signaling Technology, 9953S), and arbidol-HCl (Selleckchem, S2120). Calpain Inhibitor set includes ALLN, calpain inhibitor III, calpeptin, and E-64d used in the viral inhibition assays (208733-1SET, Sigma-Aldrich).

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin/streptomycin and Glutamax (Gibco) at 37 °C with 0.05 CO2. Transfection was performed in triplicate using a 96-well Shuttle nucleofector (Lonza). Briefly, 250 ng of plasmid per 2 × 10⁵ cells was transfected using Amaxa solution SF (Lonza) and program CA-138. Three days after transfection, cells were then incubated in DMEM containing 0.75 mM IBMX (3-isobutyl-1-methylxanthine; Enzo life sciences) for 15 min. The cells were next treated with 6.67 nM of BMN 111 (CNP)^{52 (link)} and incubated for another 15 min. The reaction was terminated with 40 μl of lysis solution, and the cGMP concentration was measured by a competitive enzyme immunoassay (Molecular Devices, R8075). Luciferase levels were measured using the ONE-Glo Assay (Promega) and the results are presented as the cGMP/luciferase normalized to the wild-type plasmid transfected cells. For the dose-response curves, the CNP concentrations assayed in triplicate were 33.33, 6.67, 1.33, 0.27, 0.053, 0.0107, and 0.002133 nM. The cGMP concentrations were assayed as above and cellular concentrations calculated assuming 1.6E5 cells/well with a diameter of 35 μm.