*2.6. Alkaline Phosphatase Activity*

SaOS-2 cells seeded in six-well plates during di fferentiation in the presence or not of the various treatments, as described above, were collected in Cytobuster Protein Extraction Reagent (Milipore, Burlington, MA, USA). After sonication twice on ice and centrifugation at 4 ◦C for 15 min at 1000× *g*, alkaline phosphatase (ALP) activity was measured in the supernatants using the QuantiFluo Alkaline Phosphatase Assay Kit following the manufacturer's instructions. The ALP activity was normalized to protein content for each well, and data were expressed as percentages relative to the control values.

#### *2.7. Western Blot Analysis of RUNX and RUNX-2 Phosphorylation*

Western blot analysis was performed in SaOS-2 cells six days after di fferentiation and treated or not (control) as described above. Whole-cell lysates and nuclear extracts were obtained as previously described in References [13,33], respectively. Equal amounts of nuclear proteins were then incubated with antibody against Runt-related transcription factor 2 (RUNX-2) for 1 h at 4 ◦C. Subsequently, the immune complexes were precipitated using Protein A/G PLUS-Agarose. The immunoprecipitates (200 μg) were mixed with Laemmli bu ffer for 5 min at 95 ◦C, subjected to SDS/PAGE, and electrotransferred to a PVDF membrane [13]. Phospho-RUNX-2 (p-RUNX-2), RUNX-2, histone H3, and β-actin were visualized using antibody anti-phospho-tyrosine proteins, anti-RUNX-2, anti-histone H3, or anti-β-actin, respectively. Antigen–antibody complexes were detected using chemiluminescence ECL Western Blotting Detection Reagent kit. Digital images of the bands were detected by Amersham Imager A600 (GE Healthcare, Chicago, IL, USA).

## *2.8. Alizarin Red S Assay*

The deposition of calcium was measured 12 and 24 days after di fferentiation in cells treated as described above. Cells were fixed in 4% paraformaldehyde for 15 min after washing twice with NaCl/Pi for a few minutes; subsequently, they were washed another three times with deionized water. Calcium mineral deposits were stained by using 2% Alizarin Red S at pH 7.8 for 2 min and were destained using 10% cetylpyridinium chloride in deionized water for 60 min at 50 ◦C. The absorbance of Alizarin Red S extracts was measured at 560 nm. Calcium content was evaluated using a standard curve of hydroxyapatite (100 μg/mL in cetylpyridinium chloride solution) and expressed as mg hydroxyapatite (HA) per cm2.
