Fig. 8.

Slc39a8 participates in the regulation of iron overload during the calcification of VSMCs. VSMCs were transfected with siRNA against mouse Slc39a8 for eight hours and then switched to complete DMEM/F12 medium; knockdown efficiency was assessed by qPCR 48 h after transfection (A). Total cellular proteins were used to determine the expression of Runx2 and FTH1 by western blotting; *P < 0.05 and **P < 0.01 (B and C). Alizarin red staining was performed to assess calcium deposition (D). ALP activity in VSMCs was measured; *P < 0.05, **P < 0.01 (E). The levels of malondialdehyde (MDA) were also assessed (F). Phen Green SK was used to evaluate intracellular Fe²⁺. C11-BODIPY 581/591 was employed to analyse the fluorescent cytosolic and lipid ROS in VSMCs. Scale bar: 50 µm (G and H). Following this, the cells were switched to calcification medium (CM) or CM with or without ferric ammonium citrate (FAC), and ALP activity in VSMCs was measured again; *P < 0.05, and **P < 0.01 (I). Total cellular protein was used to determine the expression of Runx2 and FTH1 by western blotting. *P < 0.05, and **P < 0.01 (J‒L). Alizarin red staining was performed to determine calcium deposition (M). The levels of malondialdehyde (MDA) were assessed (N)