2.5. Cyclic GMP Measurement

rSMC were seeded on 24-well plates and cultured for 72 h in the presence of 1 or 3 mM Mg, combined or not with sirolimus (2 and 50 ng/mL). The cells were then treated for 30 min with Sodium Nitroprusside (SNP 10 µM) (Sigma Aldrich, St. Louis, MO, USA), a donor of nitric oxide (NO). The Cyclic GMP XP Assay Kit (Cell Signaling, Danvers, MA, USA) was utilized according to the manufacturer’s instructions. The percentage of activity was calculated as follows: % activity = 100 × [(A − Abasal)/(Amax − Abasal)], where A is the sample absorbance, Amax is the absorbance at maximum stimulation (high cyclic GMP concentration), and Abasal is the absorbance at basal level (no cGMP). The experiment was performed 3 times in triplicate.