Fig. 4.

Iron overload promotes the osteogenic differentiation of VSMCs. Phen Green SK was used to evaluate intracellular Fe²⁺ in VSMCs. Scale bar: 50 µm (A and B). VSMCs were cultured in GM or CM with or without various concentrations of FAC (25, 50, 100 µM) for 14 days to detect osteogenic indicators. Alizarin red staining and quantitative analysis were conducted to determine calcium deposition. White arrow head indicates the calcified deposits. Scale bar: 100 µm (C and D). The expression of Runx2, BMP2, FTL, and FTH1 was determined by western blot and quantified by densitometry. *P < 0.05, **P < 0.01 (E‒G). ALP activity in VSMCs was measured; *P < 0.05, **P < 0.01 (H)