Alkaline phosphatase activity and mineralization

Alkaline phosphatase (ALP) activity was measured using a commercially available kit (LabAssay^™ ALP kit, Wako Pure Chemicals, Tokyo, Japan) according to standard procedures. After 3, 7, and 14 days of fluvastatin treatment, the samples were subsequently detached using a cell scraper and sonicated on ice (Branson, MO, USA) (n = 5). Cell debris was removed by centrifugation at 15,000 rpm. ALP activity levels were normalized against total protein using a protein assay reagent (BCA, Pierce Chemical, Rockford, IL, USA). After 14 days of fluvastatin treatment, cells were fixed in 10% neutral buffered formalin. ALP substrate solution (Roche Diagnostics, Basel, Switzerland) was added to fixed cells for staining. Cells were subsequently washed with PBS and images were recorded.

After 21 days of fluvastatin treatment, cells were fixed in 10% neutral buffered formalin and then stained with Alizarin Red S solution (Wako Pure Chemical Industries Ltd., Osaka, Japan) for 5 min at room temperature. Cells were washed with PBS and images were recorded.