(A–C) Scatter dot plots and arithmetic means ± SEM (n=6; arbitrary units, a.u.) of MSX2 (A), CBFA1 (B) and ALPL (C) relative mRNA expression in HAoSMCs following treatment for 24 h with control (CTR), C2-ceramide (C2-cer) or β-glycerophosphate (Pi) alone and together with C2-ceramide (C2-cer). (D) Scatter dot plots and arithmetic means ± SEM (n=10, a.u.) of normalized ALP activity in HAoSMCs following treatment for 7 days with control (CTR), C2-ceramide (C2-cer) or β-glycerophosphate (Pi) alone and together with C2-ceramide (C2-cer). (E) Representative images showing Alizarin Red staining in HAoSMCs following treatment for 11 days with control (CTR), C2-ceramide (C2-cer) or calcification medium (Calc.) alone and together with C2-ceramide (C2-cer). The calcified areas are shown as red. (F) Scatter dot plots and arithmetic means ± SEM (n=4; µg/mg protein) of calcium content in HAoSMCs following treatment for 11 days with control (CTR), C2-ceramide (C2-cer) or calcification medium (Calc.) alone and together with C2-ceramide (C2-cer). *(P<0.05), **(P<0.01), ***(P<0.001) significant compared with control HAoSMCs; †(P<0.05), ††(P<0.01), †††(P<0.001) significant compared with Pi/Calc.-treated HAoSMCs.