Fig 1: Mg2+ prevents Pi-induced calcification in hVSMCs. (A) Alizarin red staining was negative in Mg2+-supplemented Pi cultures, indicating the absence of calcification after 2, 4 and 8 days. (B) Ca2+ deposition on VSMCs was increased in Pi-treated cells compared with controls, but was absent after 2.0 mmol/L Mg2+ supplementation. (C) Pi-induced calcification decreased ALP and non-significantly decreased calcification inhibitor MGP and VSMC contractile genes SM22a and CNN1 after 6 days (black bars) and 8 days (white bars) of incubation, which was prevented by Mg2+. (D) Protein expression of OPN, SM22a and MGP was measured and quantified. Data are shown as fold changes compared with the day-matched controls and presented as the mean of three separate experiments, consisting of three replicate cultures ± SD. *P < 0.05 versus day-matched control.