Fig. 4.

The mRNA expression profile and activity of SKs and SMases during VSMC matrix mineralization. VSMCs were cultured in control or osteogenic (Osteo) medium. At day 0 (D0), early, mid, and late mineralization cell lysates were prepared for activity assays or total RNA was extracted for quantification of target genes by qPCR. SPHK1 and SPHK2 expression, SK activity (A); SMPD1, SMPD2, and SMPD4 expression (B); and L-SMase and nSMase activity (C). mRNA levels are shown relative to the housekeeping genes, PP1A and RPL12, as detailed in the Materials and Methods. The data are mean ± SEM from eight independent experiments. *P < 0.05 control versus osteogenic by two-way ANOVA with Dunnett’s multiple comparisons post test.