(A–D) Scatter dot plots and arithmetic means ± SEM (n=11; arbitrary units, a.u.) of Sgk1 (A), Msx2 (B), Cbfa1 (C) and Alpl (D) relative mRNA expression in MAoSMCs isolated from Asm^+/+ and Asm^−/− mice and following treatment for 24 h with control (CTR) or β-glycerophosphate (Pi) without or with bacterial sphingomyelinase (SMase). (E) Representative images showing Alizarin Red staining in MAoSMCs isolated from Asm^+/+ and Asm^−/− mice and following treatment for 11 days with control (CTR) or calcification medium (Calc.). The calcified areas are shown as red. (F) Scatter dot plots and arithmetic means ± SEM (n=8; µg/mg protein) of calcium content in MAoSMCs isolated from Asm^+/+ and Asm^−/− mice and following treatment for 11 days with control (CTR) or calcification medium (Calc.) without or with bacterial sphingomyelinase (SMase). *(P<0.05), **(P<0.01), ***(P<0.001) significant compared with control Asm^+/+ MAoSMCs; ††(P<0.01), †††(P<0.001) significant between Pi/Calc.-treated Asm^+/+ and Asm^−/− MAoSMCs; §(P<0.05), §§(P<0.01), §§§(P<0.001) significant between Pi/Calc.- and Pi/Calc.+SMase-treated Asm^−/− MAoSMCs.