Alkaline phosphatase (ALP) activity, Alizarin red staining, Adipored assay and Oil red staining

To measure the degree of osteogenesis, the transduced hBMSCs on the 12-well cell culture plate were grown in the culture medium until approximately 100% confluence, and then the growth medium was replaced with osteogenic medium (Sigma, MO) containing 10 mM β-glycerophosphate, 50 µg/mL L-ascorbic acid, and 100 nM dexamethasone, supplemented with and without phenamil (20 μM) (Sigma) and/or BMP-2 (100 ng/ml) (R&D System, MN). At day 3 of osteoinduction, the cells were fixed with 10% formalin (Sigma), stained with ALP colorimetric assay kit (Sigma) containing Nitro Blue tetrazolium, 5-Bromo-4-chloro-3-indoxylphosphate and AP buffer (100 mM Tris pH 8.5, 100 mM NaCl, 50 mM MgCl₂), and imaged with Olympus BX 51 microscope (Japan). To further assess ALP activity, the cells were digested in 0.2% NP-40 lysis buffer (Life technologies, CA), subsequently incubated in the buffer containing p-nitrophenol phosphate substrate (Sigma), and measured of the absorbance at 405 nm using a multi-plate reader. Each measurement was finally standardized with total DNA content detected by PicoGreen dsDNA Assay (Life technologies). Alizarin red staining was performed to detect calcium deposition of cells after 2 weeks of osteogenic induction. The fixed cells with 10% formalin, were then stained with 2% alizarin red solution (Sigma) for 5 mins, and visualized with an Olympus BX 51 microscope. The stained cells were further quantitatively analyzed through dissolving the stained cells in 10% (v/v) acetic acid followed by measurement of the absorbance at 405 nm.

To analyze adipogenic differentiation, the transduced cells in 12-well plate at approximately 100% confluence were incubated in human MesenCult™ adipogenic medium (Lonza). Intracellular lipid accumulation within the differentiated adipocytes was assessed after 1 week of post-induction using the Adipored kit (Lonza) by absorbance at 572 nm. For visualization of extracellular lipid expression, the cells at week 2 post-induction were stained with Oil Red O solution (Sigma) for 15 mins and were imaged with the microscope.