Alp substrate solution

Following the osteogenic differentiation experiments, the iPSCs were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min at room temperature, and washed twice more with distilled water. The fixed cells were stained with an ALP substrate solution (Roche Diagnostics, Basel, Switzerland) for 30 min at room temperature. The cells were then washed three times with distilled water, and images were captured using a phase-contrast microscope.

Three days after treatment, cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 5 min at room temperature, and then washed three more times with PBS. For staining, an ALP substrate solution (Roche Diagnostics, Basel, Switzerland) was added to fixed cells for 30 min at room temperature. Cells were then washed three times with distilled water, and images were scored. ALP activity was quantitatively measured as follows. The cells were washed twice with PBS and lysed with lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, complete protease inhibitor mixture, and 1% Nonidet P-40). The protein concentration was measured with a DC protein assay kit (Bio-Rad, Marnes-la-Coquette, France) according to the manufacturer's instructions. ALP activity was assayed using p-nitrophenyl phosphate as a substrate, and calculated as micromoles of p-nitrophenol/min/mg of protein.
To detect calcium deposits in mineralized tissue, cells were fixed by the same method described above and then stained with Alizarin Red S solution (pH 6.38; Wako Pure Chemical Industries Ltd., Osaka, Japan) for 5 min at room temperature.

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.