Fig. 3.
OPN phosphorylation status controls in vitro endothelial cell migration and in vitro smooth muscle cell calcification. A: Wound healing assay of confluent HUVECs in the presence or absence of recombinant OPN (50 ng/ml) or phosphorylated OPN (50 ng/ml) analyzed after 24 h.Magnification 4×. B: Bargraph representation of endothelial cell migration. The results of counted migrated cells were representative of three experiments each done in triplicate; P < 0.01. C: In vitro calcification of confluent smooth muscle cells treated with calcification medium (MEM containing 3 mM phosphate buffer) in the presence or absence of OPN (50 ng/ml) or (p)OPN (50 ng/ml). D: At day 8 of treatment, cells were washed twice with PBS and incubated overnight in 0.6 N HCl at 4°C. Calcium was estimated using the calcium assay reagent (Biovision, Mountain View, CA). Amount of total calcium is expressed as calcium mg/ml of medium. Pictures were taken at days 0 and 8 at 4× magnification. The results are representative of three independent experiments each done in triplicate; P < 0.01.