Adenosine 3 5 cyclic monophosphate sodium salt monohydrate

1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), 3-isobutyl-1-methylxanthine (IBMX), adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate, C-type natriuretic peptide (CNP), diethylamine NONOate diethylammonium salt (DEA), forskolin, guanosine 3′,5′-cyclic monophosphate sodium salt, Polybrene, Tris base, and vardenafil, were from Sigma-Aldrich (Burlington, MA, USA). Dulbecco’s modified Eagle medium (DMEM), Medium 199 (M199), L-glutamine, and penicillin–streptomycin (P/S) were from Gibco (Waltham, MA, USA). Foetal bovine serum (FBS) was from Scientifix (Melbourne, VIC, Australia). EGM-2 endothelial cell growth medium-2 BulletKit was from Lonza (Basel, Switzerland). Smooth muscle cell growth supplement was from ScienCell (Carlsbad, CA, USA). Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) was from ChemSupply (Adelaide, SA, Australia). Coelenterazine h was from Nanolight (Pinetop, AZ, USA).

To determine RNA stabilities for sRNAs in different mutants constructed in the fur⁺ background (Supplemental Table S1) all cultures were grown to exponential phase. RyhB or MicA expression were induced from a ryhB promoter by the addition of dipyridyl (iron chelator) while CyaR expression was induced from its native promoter by the addition of cyclic AMP (Adenosine 3′, 5′- cyclic monophosphate sodium salt monohydrate, Sigma) at a 5 mM final concentration. Under exponential growth conditions expression of MgrR, GcvB, and ChiX is constitutive. After 15 min of sRNA expression a culture sample (T₀) was collected. Following that, rifampicin was added to inhibit all further transcription and additional samples were collected 1, 2, 4, and 6 min after rifampicin addition. To determine RyhB stability in mutants constructed in a fur deletion background strains were grown in MOPS EZ rich defined medium to exponential phase and a culture sample was collected (T₀). Rifampicin was added to all cultures to inhibit transcription and additional samples were collected at 2, 5, 10, 15, and 30 min after rifampicin addition. All samples were subjected to RNA extraction as described above.