Time course expression of osteogenesis-related markers in valvular interstitial cells (VIC) exposed for 2, 7, 14 and 21 days to C (lane 1), high glucose concentration (HG) (lane 2), osteogenic medium (OM) (lane 3) or a combination of HG and OM (HGOM) (lane 4). The level of protein expression of α-smooth muscle actin (α-SMA) (A), runt-related transcription factor 2 (Runx2) (B), alkaline phosphatase (ALP) (C) and bone sialoprotein (BSP) (D) was determined by Western blot for different activation conditions, quantified by reporting to β-actin level at different periods and normalized to the day 2 values. Inserts show the level of protein expression at day 2 expressed as fold induction over control cells exposed to 5 mM glucose. Results represent the means ± S.D. of three independent experiments performed in duplicate (n = 6). Representative blots are shown above the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001 versus values determined at day 2. & p < 0.05, && p < 0.01. &&& p < 0.001 versus values determined at day 7; Inserts: * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.